TITLE: iconHi-C Protocol (ver. 1.0)
AUTHORS: Mitsutaka Kadota, Osamu Nishimura, Hisashi Miura, Kaori Tanaka, Ichiro Hiratani, Shigehiro Kuraku

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Hi-C, a derivative of chromosome conformation capture (3C) targeting the whole genome, was originally developed as a means for characterizing chromatin conformation and is recently employed widely in scaffolding nucleotide sequences obtained by </span><span style = "font-style:italic;">de novo</span><span> genome sequencing. To gain insights into the best practice of Hi-C scaffolding, we performed a multifaceted methodological comparison and optimized various factors during sample preparation, sequencing, and computation. As a result, we have identified some key factors for efficient Hi-C data acquisition, including preparation of tissues, library preparation conditions, and quality controls of prepared samples. By incorporating those optimal conditions revealed by our experience, we release this customized protocol designated the ‘inexpensive and controllable Hi-C (iconHi-C) protocol’.</span></div></div>

[STEPS]
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Dissociate cells, count cell number, and collect 1 ×107 cells in a microtube (1.5 or 2.0 ml).
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Centrifuge the cells.Centrifugation speed: Centrifugation time: Centifugation temperature:
4 °C
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Follow sample preparation procedure that matches the sample type; dissociated cells (e.g., cultured cells and nucleated red blood cells), fibrous tissue (e.g. muscle, kidney, intestine, heart), or soft tissue (e.g., liver, brain, testis, and embryonic tissues).
?. [Section 2. (DAY 0) Fixation of cells]
Take the sample tube out from the ice bucket (from Step 5) and add -.
1 ml
[ ice-cold fixing solution]
?. [Section 2. (DAY 0) Fixation of cells]
Prepare the fixing solution and put on ice.  ABC1ReagentAmountFinal Concentration216% formaldehyde solution1 ml(1%)3PBS (-)15 ml　4Total16 ml
ABC1ReagentAmountFinal Concentration216% formaldehyde solution1 ml(1%)3PBS (-)15 ml　4Total16 ml
Make fresh every time.
?. [Section 2. (DAY 0) Fixation of cells]
Incubate the cell-suspension for  in a heat block or a water bath set at .
25 °C
?. [Section 2. (DAY 0) Fixation of cells]
Quick spin the sample tube.
?. [Section 2. (DAY 0) Fixation of cells]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 2. (DAY 0) Fixation of cells]
Resuspend the pellet in - and aliquot the cell-suspension into microtubes (1.5 ml) at 1-2 ×106 cells/tube.
1 ml
[ice-cold PBS]
?. [Section 2. (DAY 0) Fixation of cells]
Centrifuge the sample tubes.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Dissect and collect tissue in a microtube (1.5 or 2.0 ml) and freeze immediately in liquid nitrogen.
Store the frozen tissue in an ultra-low temperature freezer (e.g.,  ).
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Pre-cool a mortar and a pestle in an ultra-low temperature freezer (e.g., ) for at least .
-80 °C
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Pour liquid nitrogen into the pre-cooled mortar with a pestle.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Transfer tissue (up to 1 cm3 in size) into the mortar and grind the tissue until it becomes a fine powder.
Pour liquid nitrogen into the mortar occasionally, to keep the motar and the pestle cold during the grinding process.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Resuspend the powderized tissue in liquid nitrogen and transfer the suspension into a 50 ml tube pre-cooled with liquid nitrogen.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Close the screw cap loosely.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Proceed immediately to cell fixation (Step 11) or store the powderized tissue in an ultra-low temperature freezer (e.g., ) until use.
-80 °C
?. [Section 2. (DAY 0) Fixation of the tissue]
Prepare fixing solution and put on ice.  ABC1ReagentAmountFinal Concentration216% formaldehyde solution1 ml(1%)3PBS (-)15 ml　4total16 ml
ABC1ReagentAmountFinal Concentration216% formaldehyde solution1 ml(1%)3PBS (-)15 ml　4total16 ml
Make fresh every time.
?. [Section 2. (DAY 0) Fixation of the tissue]
Take the sample tube out from the ultra-low temperature freezer (from Step 10) and immediately add -.
10 ml
[ice-cold fixing solution]
?. [Section 2. (DAY 0) Fixation of the tissue]
Incubate the tissue suspension for - in a heat block or a water bath set at .
25 °C
?. [Section 2. (DAY 0) Fixation of the tissue]
Quick spin the sample tube.
?. [Section 2. (DAY 0) Fixation of the tissue]
Centrifuge the sample tube.  Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 2. (DAY 0) Fixation of the tissue]
Resuspend the pellet in - and aliquot the tissue-suspension evenly into five to ten microtubes (1.5 ml).
1 ml
[ ice-cold PBS]
Aliquot a 4 mm3-size tissue into five to ten microtubes.Adjust the number of microtubes proportionally for a tissue at different size.
?. [Section 2. (DAY 0) Fixation of the tissue]
Centrifuge the sample tubes.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Pre-cool the Tokken stainless-steel tubes and bullets in liquid nitrogen.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Transfer tissue (up to 4 mm3 in size) into each stainless-steel tube.
Break a large tissue into small pieces if necessary.Do not let the tissue thaw during the powderization process.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Put a bullet in the stainless-steel tube and close the screw cap.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Assemble the Tokken tube holder with three stainless-steel tubes.
The tube holder should always be assembled with three stainless-steel tubes to balance the weight.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Submerge the tube holder (with tubes) in liquid nitrogen and wait ~.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Take out the tube holder (with tubes) from liquid nitrogen and immediately place inside the Tokken acryl-grinder, then vigorously shake the grinder for about 100 strokes to powderize the tissue.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Remove the bullet and collect the powderized tissue in a microtube (1.5 or 2.0 ml) pre-cooled in liquid nitrogen.
Hand warm the lid of the deep-frozen microtube before closing its lid; otherwise the tube may break.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Proceed immediately to cell fixation (Step  11) or store the powderized tissue in an ultra-low temperature freezer (e.g., ) until use.
-80 °C
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Prepare the fixing solution and put on ice.  ABC1ReagentAmountFinal Concentration216% formaldehyde solution1 ml(1%)3PBS (-)15 ml　4total16 ml
ABC1ReagentAmountFinal Concentration216% formaldehyde solution1 ml(1%)3PBS (-)15 ml　4total16 ml
Make fresh every time.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Take a sample tube (microtube) out from the ultra-low temperature freezer, immediately add -.
1 ml
[ice-cold fixing solution]
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Incubate the tissue-suspension for - in a heat block or a water bath set at .
25 °C
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Quick spin the sample tube.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Centrifuge the sample tube. Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Resuspend the pellet in - and aliquot the tissue-suspension evenly into five to ten microtubes (1.5 ml).
1 ml
[ice-cold PBS]
Aliquot a 4 mm3-size tissue into five to ten microtubes.Adjust the number of microtubes proportionally for a tissue at different size.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Centrifuge the sample tubes.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Remove the supernatant.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Put the tube on ice, and proceed immediately to fixation (Step 6).
?. [Section 2. (DAY 0) Fixation of cells]
Vortex until the cells are fully resuspended in fixing solution.
?. [Section 2. (DAY 0) Fixation of the tissue]
Vortex until the tissue powder is fully resuspended in the fixing solution.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Place the tube in an ultra-low temperature freezer (e.g., ).
-80 °C
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Wait until the liquid nitrogen evaporates completely.
?. [Section 2. (DAY 0) Fixation of the tissue]
Vortex mix.
?. [Section 2. (DAY 0) Fixation of the tissue]
Put the sample tube on ice for ~ .
?. [Section 2. (DAY 0) Fixation of the tissue]
Remove the supernatant.
?. [Section 2. (DAY 0) Fixation of the tissue]
Add . (1/2)
[ice-cold PBS (-)]
?. [Section 2. (DAY 0) Fixation of the tissue]
Vortex mix. (1/2)
?. [Section 2. (DAY 0) Fixation of the tissue]
Centrifuge the sample tube.  (1/2)Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 2. (DAY 0) Fixation of the tissue]
Remove the supernatant. (1/2)
?. [Section 2. (DAY 0) Fixation of the tissue]
Remove the supernatant.
?. [Section 2. (DAY 0) Fixation of cells]
Add 1/20 volume of a  to quench the formaldehyde.
[glycine solution]
?. [Section 2. (DAY 0) Fixation of cells]
Vortex mix.
?. [Section 2. (DAY 0) Fixation of cells]
Put the sample tube on ice for ~.
?. [Section 2. (DAY 0) Fixation of cells]
Remove the supernatant.
?. [Section 2. (DAY 0) Fixation of cells]
Add . (1/2)
[ ice-cold PBS (-)]
?. [Section 2. (DAY 0) Fixation of cells]
Vortex mix. (1/2)
?. [Section 2. (DAY 0) Fixation of cells]
Centrifuge the sample tube. (1/2)Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 2. (DAY 0) Fixation of cells]
Remove the supernatant. (1/2)
?. [Section 2. (DAY 0) Fixation of cells]
Remove the supernatant.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
The pre-determination section quantitates the amount of DNA contained in a cell/tissue pellet prepared in the previous section. Pre-determination should always be performed for tissue samples, but it is optional when the DNA content in a pellet is estimable,. e.g., for cultured cells and nucleated red blood cells.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Dissect and collect tissue in a microtube (1.5 or 2.0 ml) and freeze immediately in liquid nitrogen.
Store the frozen tissue in an ultra-low temperature freezer (e.g.,  ).
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Vortex mix until the tissue powder is fully resuspended in the fixing solution.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Vortex mix.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Put the sample tube on on ice for ~ .
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Remove the supernatant.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Add .  (1/2)
[ice-cold PBS (-)]
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Vortex mix. (1/2)
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Centrifuge the sample tube. (1/2) Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Remove the supernatant. (1/2)
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Remove the supernatant.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the permeabilization buffer 1 (PB1).  ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 8.0)400 μl(10 mM)35 M NaCl80 μl(10 mM)410% (w/v) NP-40800 μl(0.2%)5H2O38.72 ml 6Total40 ml
This section includes the following steps.Cell/tissue permeabilization - Steps 26 - 51Restriction enzyme digestion - Steps 52 - 54
The number of cells to use for Hi-C is determined based on the amount of DNA.Use 1×106  cells for an animal with the genome size of 3-3.5 Gb (e.g., human and mouse), or 2×106 cells for an animal with the genome size of 1-1.5 Gb (e.g., chicken and western clawed frog).For tissue samples, use a pellet that contains 2-10 μg of DNA.
Follow cells/tissue resuspension steps as written, i.e., vortex mix or pipet mix.
ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 8.0)400 μl(10 mM)35 M NaCl80 μl(10 mM)410% (w/v) NP-40800 μl(0.2%)5H2O38.72 ml 6Total40 ml
Filtrate and store at  .
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take ( ×n) + 5% extra volume of PB1 in a new tube, add 1/100 vol of a proteinase inhibitor cocktail (PI), and put on ice.
400 µl
Add PI to PB1 just before use.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take the frozen cells out from the freezer (from Step 24) and immediately add .
[PB1 (with PI)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Vortex mix.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate the sample tube on ice for  with periodical mixing every -.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-1000 pipet.
300 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 or a P-200 pipet, and put the sample tube on ice.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
.Prepare the permeabilization buffer 2 (PB2) at .  ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 2.125 μl(1X)310% (w/v) SDS7.5 μl(0.3%)4PI (100X)2.5 μl(1X)5H2O215 μl 6Total250 μl
0 Room temperature
ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 2.125 μl(1X)310% (w/v) SDS7.5 μl(0.3%)4PI (100X)2.5 μl(1X)5H2O215 μl 6Total250 μl
Make fresh every time.Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer 2.1]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10% (w/v) SDS]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[PI (100X)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  in a tube of appropriate size.
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and pipet mix.
[PB2]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate/shake the sample tube in a thermal mixer for  at , .
37 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube.
[20% TritonX-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate/shake the sample tube in a thermal mixer for  at , .
37 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the DNA-preparation buffer .   ABC1ReagentAmountFinal Concentration25 M NaCl250 μl(250 mM)30.5 M EDTA50 μl(5 mM)410% (w/v) SDS500 μl(1%)5H2O4.2 ml 6Total5 ml
ABC1ReagentAmountFinal Concentration25 M NaCl250 μl(250 mM)30.5 M EDTA50 μl(5 mM)410% (w/v) SDS500 μl(1%)5H2O4.2 ml 6Total5 ml
Store DNA-preparation buffer at  .Warm the DNA-preparation buffer at   to dissolve precipitates formed during storage.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-200 pipet.
200 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 or a P-200 pipet, and put the sample tube on ice.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare washing buffer (WB) with NEBuffer DpnII (for the DpnII digested sample).  ABC1ReagentAmount (x1 wash cycle)Final Concentration210X NEBuffer DpnII50 μl(1X)320 mg/ml BSA2.5 μl(0.1 mg/ml)420% (w/v) TritonX-1001.25 μl(0.05%)5H2O446.25 μl 6Total500 μl
ABC1ReagentAmount (x1 wash cycle)Final Concentration210X NEBuffer DpnII50 μl(1X)320 mg/ml BSA2.5 μl(0.1 mg/ml)420% (w/v) TritonX-1001.25 μl(0.05%)5H2O446.25 μl 6Total500 μl
Do not use NEBuffer 3.1 for the samples for DpnII digestion.Make WB for HindIII digestion with NEBuffer 2.1 (that contains BSA).Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer DpnII]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20 mg/ml BSA]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20% (w/v) TritonX-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  in a tube of appropriate size.
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet, and put the sample tube on ice.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the restriction-enzyme mix (for DpnII digestion) on ice.  ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer DpnII20 μl(1X)320 mg/ml BSA1 μl(0.1 mg/ml)4DpnII (50 U/μl)8 μl(400 U)520% Triton X-1000.5 μl(0.05%)6H2O170.5 μl 7Total200 μl
ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer DpnII20 μl(1X)320 mg/ml BSA1 μl(0.1 mg/ml)4DpnII (50 U/μl)8 μl(400 U)520% Triton X-1000.5 μl(0.05%)6H2O170.5 μl 7Total200 μl
Do not use NEBuffer 3.1 for DpnII because non-specific cleavage (star activity) may be induced.Use NEBuffer 2.1 (that contains BSA) for HindIII digestion.For restriction enzymes at different concentration, e.g., HindIII at 100U/μl, adjust the volume with H2O.Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer DpnII]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20 mg/ml BSA]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[DpnII (50 U/ul)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20% Triton X-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  in a tube of appropriate size.
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and pipet mix.
[restriction-enzyme mix]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate the sample tube in a thermal mixer for ~ at , .
37 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
This section includes the following steps.DNA fill-in reaction - Steps 56 - 76Ligation reaction - Steps 77 - 79
Follow cells/tissue resuspension steps as written, i.e., vortex mix or pipet mix.
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-200 pipet.
150 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-200 pipet or a P-100 pipet, and put the sample tube on ice.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare the washing buffer (WB) with NEBuffer 2.1.  ABC1ReagentAmount (x1 wash cycle)Concentration210X NEBuffer 2.150 μl(1X)320% (w/v) TritonX-1001.25 μl(0.05%)4H2O448.75 μl 5Total500 μl
ABC1ReagentAmount (x1 wash cycle)Concentration210X NEBuffer 2.150 μl(1X)320% (w/v) TritonX-1001.25 μl(0.05%)4H2O448.75 μl 5Total500 μl
Prepare buffer to perform total of four or two wash cycles (at Steps 61, 66 and 73), for the DpnII-digested sample or the HindIII-digested sample respectively.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[10X NEBuffer 2.1]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% (w/v) TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Repeat the washing cycle (Steps 61 - 65) twice more for the DpnII-digested sample (total of three wash cycles).
Additional washing cycle is not required for the HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare the DNA fill-in mix on ice.  ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 210 μl(1X)30.4 mM biotin-14-dATP3.75 μl(15 μM)41 mM dCTP1.5 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)6 μl(30 U)820% TritonX-1000.25 μl(0.05%)9H2O75.5 μl 10Total100 μl   ABC1ReagentAmountFinal Concentration210X NEBuffer 210 μl(1X)31 mM dATP1.5 μl(15 μM)40.4 mM biotin-14-dCTP3.75 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)3 μl(15 U)820% TritonX-1000.25 μl(0.05%)9H2O78.5 μl 10Total100 μl
ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 210 μl(1X)30.4 mM biotin-14-dATP3.75 μl(15 μM)41 mM dCTP1.5 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)6 μl(30 U)820% TritonX-1000.25 μl(0.05%)9H2O75.5 μl 10Total100 μl
ABC1ReagentAmountFinal Concentration210X NEBuffer 210 μl(1X)31 mM dATP1.5 μl(15 μM)40.4 mM biotin-14-dCTP3.75 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)3 μl(15 U)820% TritonX-1000.25 μl(0.05%)9H2O78.5 μl 10Total100 μl
Make a DNA fill-in mix that contains either biotin-14-dATP or biotin-14-dCTP, for the DpnII-digested sample or the HindIII-digested sample respectively.Make 5% extra volume.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[10X NEBuffer 2]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dCTP]
Add   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dGTP]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dTTP]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[0.4 mM biotin-14-dATP]
Add   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[Klenow DNA polymerase (5 U/μl)]
Add   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Take  in a tube of appropriate size.
[H2O]
Take   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and pipet mix.
[DNA fill-in mix]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Incubate the sample tube in a thermal mixer for  at , .
25 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare ligation mix on ice.  ABC1ReagentAmountFinal Concentration2T4 DNA ligase buffer (10X)10 μl(1X)3T4 DNA ligase (2,000 U/ul)2 μl(4,000 CEU)420% TritonX-1000.25 μl(0.05%)5H2O87.75 μl 6Total100 μl
ABC1ReagentAmountFinal Concentration2T4 DNA ligase buffer (10X)10 μl(1X)3T4 DNA ligase (2,000 U/ul)2 μl(4,000 CEU)420% TritonX-1000.25 μl(0.05%)5H2O87.75 μl 6Total100 μl
Prepare T4 DNA ligase buffer in small aliquots and store them at   when the buffer is thawed for the first time.Always use an aliquot that is not thawed and frozen repeatedly.Use 2,000 CEU of T4 DNA ligase for the HindIII-digested sample.Make 5% extra volume.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[T4 DNA ligase buffer (10X)]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[T4 DNA ligase (2,000 U/ul)]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and pipet mix.
[ligation mix]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Incubate the sample tube in a thermal mixer for - at , .
16 °C
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Prepare DNA-extraction mix.  ABC1ReagentAmount (x1 reaction)Final Concentration21M Tris-HCl (pH 8.0)2 μl(10 mM)30.5M EDTA2 μl(5 mM)410% SDS30 μl(1.5%)55M NaCl15 μl(375 mM)620 mg/ml Proteinase K10 μl(1 mg/ml)7H2O141 μl 8Total200 μl
ABC1ReagentAmount (x1 reaction)Final Concentration21M Tris-HCl (pH 8.0)2 μl(10 mM)30.5M EDTA2 μl(5 mM)410% SDS30 μl(1.5%)55M NaCl15 μl(375 mM)620 mg/ml Proteinase K10 μl(1 mg/ml)7H2O141 μl 8Total200 μl
Prepare DNA-extraction mix at  .Make 5% extra volume.
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[1M Tris-HCl (pH 8.0)]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[0.5M EDTA]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[10% SDS]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[5M NaCl]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[20 mg/ml Proteinase K]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Take  in a tube of appropriate size.
[H2O]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add  to the sample tube (from Step 81) and pipet mix.
[DNA-extraction mix]
The total amount of the mixture will be  .
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add  and  to the control tubes (ctr-1 from Step 41 and ctr-2 from Step 55) and pipet mix.
[DNA-extraction mix]
[H2O]
The total amount of the mixture will be  .
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Incubate in a thermal mixer for ~ at , .
65 °C
Use a thermal mixer equipped with a heated lid to avoid condensation of water on the lid.Alternatively, the incubation can be performed in a heated oven.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Take the sample tube out from the thermal mixer (from Step 85) and let it cool down to .
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[RNase A (10 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[Proteinase K (20 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Take the sample tube out from the thermal mixer, let it cool down to , and proceed with DNA extraction.
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[Phenol/Chloroform/Isoamyl alcohol solution]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Incubate sample tube in a thermal mixer for  at , .
37 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Incubate the sample tube in a thermal mixer for  at , .
55 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Transfer ~ of the aqueous phase into a new 1.5 ml microtube.
250 µl
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube containing Phenol and mix gently.
[TE/NaCl solution]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Transfer ~ of the aqueous phase into the microtube containing the first aqueous phase.
300 µl
The total volume of the collected aqueous phase will be ~ .
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the collected aqueous phase and mix gently.
[glycogen solution (20 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the collected aqueous phase and mix gently until the solution becomes homogeneous.
[2-propanol]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Decant the supernatant, add , and mix gently to rinse the DNA pellet.
[70% EtOH]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Decant the supernatant.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Remove the supernatant completely with a P-100 or a P-200 pipet, and keep the lid open for ~ to allow the ethanol to evaporate.
Do not over dry the pellet.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add - and  to the Hi-C DNA and the control DNAs (ctr-1 and ctr-2) respectively.
30 µl
[EB]
[EB]
Mix gently to avoid shearing of the DNA.Store DNA samples at   or  .
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Quantitate DNA using  of the DNA sample with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 7. (DAY 3) Hi-C DNA QC (QC1)]
Take - of the DNA sample (ctr-1, ctr-2, and Hi-C DNA) from the previous section (Step 107) in a new PCR tube and add EB to adjust the concentration to 2-20 ng/μl.
1 µl
2 µl
The concentration of the DNA samples in a trio (ctr-1, ctr-2, and Hi-C DNA) should be within two-fold of difference, to accurately observe the size shift by Agilent Bioanalyzer or Agilent TapeStation.
?. [Section 7. (DAY 3) Hi-C DNA QC (QC1)]
Analyze  of the DpnII-digested DNA sample (ctr-2 and Hi-C DNA) using the Agilent Bioanalyzer with the DNA High Sensitivity chip, and  of the DpnII-digested DNA sample (ctr-1) using the Agilent TapeStation with the genomic tape.
1 µl
1 µl
Only ctr-2 and Hi-C DNA of the DpnII digested samples are analyzed using the Agilent Bioanalyzer with the DNA High Sensitivity chip, because ctr-1 (pre-digested DNA), >50 kb in size, exceeds the limit of the DNA size that can be analyzed. Alternatively, the DpnII-digested DNA samples can be analyzed using the Agilent TapeStation with the genomic tape.
For the HindIII-digested DNA samples, analyze   of ctr-1, ctr-2, and Hi-C DNA using Agilent TapeStation with the genomic tape.
Only qualified Hi-C DNA showing the expected pattern of size shift, i.e., reduction in ctr-2 and recovery in Hi-C DNA, with no or minimum degree of DNA degradation in ctr-1, ctr-2 and Hi-C DNA, is used for the preparation of the Hi-C library.
?. [Section 7. (DAY 3) Hi-C DNA QC (QC1)]
Store Hi-C DNA at  or proceed to the next section (Step 112).
-20 °C
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Take  -  of Hi-C DNA from the previous step (Step 111) in a 0.2 ml PCR tube and adjust the total volume to  with H2O.
250 ng
2 µg
30 µl
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Prepare T4 DNA polymerase mix on ice.  ABC1ReagentAmountFinal Concentration210X NEBuffer 2.15 μl(1X)310 mM GTP0.5 μl(100 μM)4T4 DNA pol (3 U/μl)1.67 μl(5 U)5H2O12.83 μl 6Total20 μl   ABC1ReagentAmountFinal Concentration210X NEBuffer 2.15 μl(1X)310 mM dATP0.5 μl(100 μM)410 mM dGTP0.5 μl(100 μM)5T4 DNA pol (3 U/μl)0.84 μl(2.5 U)6H2O13.16 μl7Total20 μl
ABC1ReagentAmountFinal Concentration210X NEBuffer 2.15 μl(1X)310 mM GTP0.5 μl(100 μM)4T4 DNA pol (3 U/μl)1.67 μl(5 U)5H2O12.83 μl 6Total20 μl
ABC1ReagentAmountFinal Concentration210X NEBuffer 2.15 μl(1X)310 mM dATP0.5 μl(100 μM)410 mM dGTP0.5 μl(100 μM)5T4 DNA pol (3 U/μl)0.84 μl(2.5 U)6H2O13.16 μl7Total20 μl
Make 5% extra volume.
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[10X NEBuffer 2.1]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[10 mM dGTP]
Add    and    for the T4-DNA-polymerase mix for the HindIII-digested sample.
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[T4 DNA pol (3 U/μl)]
Add   for the T4-DNA-polymerase mix for the HindIII-digested sample.
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Take  in a tube of appropriate size.
[H2O]
Take   for the T4-DNA-polymerase mix for the HindIII-digested sample.
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add  to the Hi-C DNA and mix gently.
[T4-DNA-polymerase mix]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Incubate the sample in a PCR machine for  at , and  at .
37 °C
75 °C
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Turn on the Covaris (S220 or E220) and the chiller.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Transfer the entire reaction from the previous section (Step 115) into a Covaris microTUBE and add .
[TE buffer]
The total volume will be  .
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Perform sonication Duty factor: 5%Peak incident power: 175Cycles per burst: 200Time:  ×2 Bath temperature:
7 °C
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Transfer  of the fragmented DNA into a 1.5 ml microtube.
120 µl
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Add  (×0.6 amount) of AMPure XP beads to the fragmented DNA, vortex and incubate  at .
72 µl
0 Room temperature
DNA larger than 600 bp are removed at Steps 120-122.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Transfer the supernatant into a new 1.5 ml microtube.
The supernatant contains DNA smaller than 600 bp.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Add  (×0.9 amount) of AMPure XP beads to the supernatant, vortex mix, and wait  at .
108 µl
0 Room temperature
DNA larger than 150 bp are collected.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Remove the supernatant using a P-200 pipet while the sample tube is still on the magnet.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Add  to the beads while the sample tube is still on the magnet and wait . (1/2)
[80% EtOH]
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Remove EtOH with P-200 pipet while the sample tube is still on the magnet. (1/2)
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Quick spin the sample tube, put on magnet and remove the residual EtOH completely using a P-10 or a P-20 pipet.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Air dry the beads for ~ at  with the lid kept open.
0 Room temperature
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Add  to the beads, vortex mix and incubate  at .
[EB]
0 Room temperature
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Quick spin the sample tube, put on magnet and collect the eluate in a new PCR tube.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Prepare the 2X binding-and-washing buffer (BWB).  ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 7.5)400 μl(10 mM)30.5 M EDTA (pH 8.0)80 μl(1 mM)45 M NaCl16 ml(2 M)510% Tween2080 μl(0.02%)6H2O23.44 ml 7Total40 ml
ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 7.5)400 μl(10 mM)30.5 M EDTA (pH 8.0)80 μl(1 mM)45 M NaCl16 ml(2 M)510% Tween2080 μl(0.02%)6H2O23.44 ml 7Total40 ml
Store at  .
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add .
[1 M Tris-HCl (pH 7.5)]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add .
[0.5 M EDTA (pH 8.0)]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add .
[5 M NaCl]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add .
[10% Tween20]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Take  in a tube of appropriate size.
[H2O]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Prepare 1X BWB by diluting the 2X BWB with H2O.
Store at  .
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Mix the bottle of the streptavidin beads and transfer ( × n) + 5% extra volume of beads into a new 1.5 ml microtube.
25 µl
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Put the tube on the magnetic and wait until the supernatant becomes clear.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, take off the tube from the magnet, add , and vortex mix.
[1X BWB]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, take off the tube from the magnet, add ( × n) + 5% extra volume of 2X BWB, and pipet mix.
60 µl
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add  (in 2X BWB) to the size-selected Hi-C DNA prepared in previous section (from Step 132) and vortex mix.
[beads]
The total volume will be   .
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Incubate the sample tube in a thermal mixer for  at  with periodical mixing for  at  every .
20 °C
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant while the tube is still on the magnet.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear. (1/4)
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add  and vortex mix.
[EB]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant while the tube is still on the magnet.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, take off the sample tube from the magnet, quick spin, and put the sample tube back on the magnet.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the residual EB with a P-10 or a P-20 pipet.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Resuspend the beads in  and store the sample tube at  until the next day (DAY 4).
[EB]
4 °C
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the end-repair mix on ice.  AB1ReagentAmount210X KAPA End Repair Buffer1.4 μl3KAPA End Repair Enzyme Mix1.0 μl4H2O11.6 μl5Total volume14 μl
This section consists of the following steps.End repair - Steps 157 - 175A-tailing - Steps 176 - 193Adapter ligation - Steps 194 - 212Pre-PCR (releasing DNA off the streptavidin beads) - Steps 213 - 226PCR cycle pre-determination - Steps 227 - 231Amplification of the library for Hi-C library QC (QC2) - Steps 232 - 234Library amplification - Steps 235 - 249
Library preparation is performed using the KAPA LTP DNA library kit but in a 1/5 reaction volume of the original protocol and with an additional step for PCR cycle pre-determination.
AB1ReagentAmount210X KAPA End Repair Buffer1.4 μl3KAPA End Repair Enzyme Mix1.0 μl4H2O11.6 μl5Total volume14 μl
Make 5% extra volume.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10X KAPA End Repair Buffer]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[KAPA End Repair Enzyme Mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the samle tube prepared in the previous section (from Step 156), put on magnet and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, add  to the sample tube, pipet mix, and proceed immediately to the next step.
[end-repair mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Incubate for  at  (followed by a hold at ) in a PCR machine.
20 °C
4 °C
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the A-tailing mix on ice.  AB1ReagentAmount210X KAPA A-Tailing Buffer1.0 μl3KAPA A-Tailing Enzyme0.6 μl4H2O8.4 μl5Total volume10 μl
AB1ReagentAmount210X KAPA A-Tailing Buffer1.0 μl3KAPA A-Tailing Enzyme0.6 μl4H2O8.4 μl5Total volume10 μl
Make 5% extra volume.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10X KAPA A-Tailing Buffer]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[KAPA A-Tailing Enzyme]
?. [Section 11. (DAY 4) Hi-C library preparation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the sample tube (from Step 175), pipet mix, and proceed immediately to the next step.
[A-tailing mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Incubate  at  (followed by a hold at ) in a PCR machine.
30 °C
4 °C
?. [Section 11. (DAY 4) Hi-C library preparation]
Close the lid and put on ice.
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the ligation-buffer mix (without the ligase enzyme) on ice.  AB1ReagentAmount25X KAPA Ligation Buffer2.0 μl3H2O6.0 μl4Total volume8 μl
AB1ReagentAmount25X KAPA Ligation Buffer2.0 μl3H2O6.0 μl4Total volume8 μl
Make 5% extra volume.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[5X KAPA Ligation Buffer]
?. [Section 11. (DAY 4) Hi-C library preparation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  and  to the sample tube (from Step 191) pipet mix, and put on ice.
[ligation-buffer mix]
[1 μM Illumina TruSeq compatible adapter]
In the case of multiplexed sequencing, adaptors should be balanced for their nucleotide composition at each sequence position of the index sequence.When sequencing a single library, it is better that the library is made using a mixed index adapter (e.g.,   +  ) to balance the nucleotide composition of the index sequence.We recommend using the UDI (unique dual-indexed) adapter to identify and remove index-hopped reads in the Illumina sequencing platform.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the sample tube, pipet mix, and proceed immediately to the next step.
[KAPA T4 DNA ligase]
?. [Section 11. (DAY 4) Hi-C library preparation]
Incubate for  at  (followed by a hold at ) in a PCR machine.
20 °C
4 °C
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the pre-PCR mix on ice.  AB1ReagentAmount22X KAPA HiFi Ready Mix10 μl310 μM TPC mix0.9 μl4H2O9.1 μl5Total volume20 μl
AB1ReagentAmount22X KAPA HiFi Ready Mix10 μl310 μM TPC mix0.9 μl4H2O9.1 μl5Total volume20 μl
Make 5% extra volume.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[2X KAPA HiFi Ready Mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10 μM TPC mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the sample tube (from Step 212), pipet mix, and proceed immediately to the next step.
[pre-PCR mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Put the sample tube on the magnetic and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Transfer the supernatant to a new PCR tube, add  (×1 volume) of AMPure XP beads, vortex mix, and wait  at .
20 µl
0 Room temperature
DNA larger than 200 bp will bind to the AMPure XP beads.
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant using a P-200 pipet while the sample tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads while the sample tube is still on the magnet and wait . (1/2)
[80% EtOH]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (1/2)
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and remove the residual EtOH completely using a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Air dry the beads for ~ at  with the lid kept open.
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads, vortex mix, and incubate  at .
[EB]
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and collect the eluate in a new PCR tube.
?. [Section 11. (DAY 4) Hi-C library preparation]
Perform 4 cycles of PCR amplification at 98°C 45 sec, 4 cycles of (98°C 15 sec, 60°C 30 sec, 72°C 30 sec), 72°C 1 min, and a hold at 4°C.
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the real-time PCR mix on ice  AB1ReagentAmount (x1 reaction)22X KAPA HiFi HS real-time Mix5 μl310 μM TPC mix0.35 μl4H2O3.15 μl5Total volume8.5 μl
AB1ReagentAmount (x1 reaction)22X KAPA HiFi HS real-time Mix5 μl310 μM TPC mix0.35 μl4H2O3.15 μl5Total volume8.5 μl
Make one extra reaction for the negative control that does not contain any DNA template.Make 5% extra volume.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[2X KAPA HiFi HS real-time Mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10 μM TPC mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Dispense  in a well of a PCR plate (384 well or 96 well) and add  of the pre-PCR product (from Step 224).
[real-time-PCR mix]
1.5 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Dispense  of each Fluorescence Standards (FS1~4) in the same plate in separate wells.
10 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Run real-time PCR in a ROX minus condition at, 98°C 45 sec,  20 cycles of (98°C 15 sec, 60°C 30 sec, 72°C 30 sec), 72°C 1 min.
?. [Section 11. (DAY 4) Hi-C library preparation]
Determine the threshold-PCR-cycle (Ct) that reaches Fluorescence Standard 1 (FS1) but does not exceed Fluorescence Standard 2 (FS2).
DNA for the library QC (QC2) is prepared by amplifying a small aliquot (1 μl) of the pre-PCR product with (Ct +3) cycles of PCR.Hi-C library for sequencing is prepared by amplifying the remaining 8.5 μl of the pre-PCR product with the pre-determined Ct cycle.
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the PCR-mix for Hi-C library QC (QC2) on ice.  AB1ReagentAmount22X KAPA HiFi HotStart Ready Mix5 μl310 μM TPC0.45 μl4H2O3.55 μl5Total volume9 μl
AB1ReagentAmount22X KAPA HiFi HotStart Ready Mix5 μl310 μM TPC0.45 μl4H2O3.55 μl5Total volume9 μl
Make 5% extra volume.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[2X KAPA HiFi HotStart Ready Mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10 µM TPC]
?. [Section 11. (DAY 4) Hi-C library preparation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Dispense of the PCR-mix for Hi-C library QC (QC2) in a PCR tube, and add  of the pre-PCR product (from Step 224).
9 µl
1 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Perform PCR amplification at, 98°C 45 sec, (Ct +3) cycles of (98°C 15 sec, 60°C 30 sec, 72°C 30 sec), 72°C 1 min, and a hold at 4°C.
Proceed to Step 250 in the next section after the PCR.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Dispense the restriction-mix for QC2 (with or without the RE) to a PCR tube, add - of the amplified library DNA (from Step 259), adjust the total volume of the reaction to , and incubate  at .
10 ng
20 ng
10 µl
37 °C
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Add  (×1.8 volume) of AMPure XP beads, vortex mix, and wait  at .
18 µl
0 Room temperature
DNA larger than 100 bp will bind to the AMPure XP beads.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Remove the supernatant using a P-200 pipet while the sample tube is still on the magnet.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Add  to the beads while the sample tube is still on the magnet and wait . (1/2)
[80% EtOH]
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (1/2)
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Quick spin the sample tube, put on magnet and remove the residual EtOH completely using a P-10 or a P-20 pipet.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Air dry the beads for ~ at  with the lid kept open.
0 Room temperature
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Add  to the beads, vortex mix, and incubate  at .
[EB]
0 Room temperature
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Quick spin the sample tube, put on magnet and collect the eluate in a new PCR tube.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Analyze the size difference between the RE(-) and the RE(+) samples using  of DNA with the Agilent TapeStation using the High Sensitivity D1000 tape.
2 µl
Place RE(-) and RE(+) samples for the same library side-by-side for easy comparison.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Prepare lysis buffer.  ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl300 μl(300 mM)40.5 M EDTA (pH 8.0)50 μl(5 mM)520% (w/v) SDS250 μl(1%)6H2O4.35 ml7Total5 ml
This section includes the following steps.Cell/tissue lysis - Steps 30 - 38DNA extraction and quantification - Steps 39 - 57
The pre-determination section quantitates the amount of DNA contained in a cell/tissue pellet prepared in the previous section. Pre-determination should always be performed for tissue samples, but it is optional when the DNA content in a pellet is estimable,. e.g., for cultured cells and nucleated red blood cells.
Follow cells/tissue resuspension steps as written, i.e., gently mix or pipet mix.
ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl300 μl(300 mM)40.5 M EDTA (pH 8.0)50 μl(5 mM)520% (w/v) SDS250 μl(1%)6H2O4.35 ml7Total5 ml
Store lysis buffer at  .Warm the lysis buffer at   to dissolve precipitates formed during storage.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and pipet mix.
[Proteinase K (20 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and mix gently.
[RNase A (10 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Incubate the sample tube in a thermal mixer for at , .
37 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and mix gently.
[Proteinase K (20 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Incubate the sample tube in a thermal mixer for  at , .
55 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Take the sample tube out from the thermal mixer, let it cool down to , and proceed with DNA extraction.
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and mix gently.
[Phenol/Chloroform/Isoamyl alcohol solution]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Transfer ~ of the aqueous phase into a new 1.5 ml microtube.
250 µl
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  ( pH 8.0, , ) to the sample tube containing Phenol and mix gently.
[TE/NaCl solution]
[Tris-HCl]
[NaCl]
[EDTA]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Transfer ~of the aqueous phase into the microtube containing the first aqueous phase.
300 µl
The total volume of the collected aqueous phase will be ~ .
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the collected aqueous phase and mix gently.
[glycogen solution (20 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add to the collected aqueous phase and mix gently until the solution becomes homogeneous.
[2-Propanol]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Decant the supernatant, add , and mix gently to rinse the DNA pellet.
[70% EtOH]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Decant the supernatant.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Remove the supernatant completely with a P-100 or a P-200 pipet, and keep the lid open for ~ to allow the ethanol to evaporate.
Do not over dry the pellet.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Dissolve the DNA in -.
30 µl
[TE]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Determine the amount of fixed tissue to use for Hi-C.
The amount of fixed tissue to use for Hi-C is a pellet/pellets that contains  -  of DNA.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the permeabilization buffer 1 (PB1).  ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 8.0)400 μl(10 mM)35 M NaCl80 μl(10 mM)410% (w/v) NP-40800 μl(0.2%)5H2O38.72 ml 6Total40 ml
This section includes the following stepsCell/tissue permeabilization - Steps 58 - 81Restriction enzyme digestion - Steps 82 - 84
The number of cells to use for Hi-C is determined based on the amount of DNA. Use 1×106  cells for an animal with the genome size of 3-3.5 Gb (e.g., human and mouse), or 2×106 cells for an animal with the genome size of 1-1.5 Gb (e.g., chicken and western clawed frog).  For tissue samples, use a pellet that contains 2-10 μg of DNA.
Follow cells/tissue resuspension steps as written, i.e., vortex mix or pipet mix.
ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 8.0)400 μl(10 mM)35 M NaCl80 μl(10 mM)410% (w/v) NP-40800 μl(0.2%)5H2O38.72 ml 6Total40 ml
Filtrate and store at  .
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[1 M Tris-HCl (pH 8.0)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[5 M NaCl]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10% (w/v) NP-40]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Filtrate and store at .
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take ( ×n) + 5% extra volume of PB1 in a new tube, add 1/100 vol of a proteinase inhibitor cocktail (PI), and put on ice.
400 µl
Add PI to the PB1 just before use.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take the frozen cells/tissue out from the freezer (from Step 28), immediately add .
[PB1 (with PI)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Vortex mix.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Homogenize the tissue by ~20 strokes of homogenization in a douncer with the tight pestle B on ice, and transfer the homogenate into a 1.5 ml microtube.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-1000 pipet.
300 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 or a P-200 pipet.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the permeabilization buffer 2 (PB2) at .  ABC1ReagentAmountFinal Concentration210X NEBuffer 225 μl(1X)320 mg/ml BSA1.25 μl(0.1 mg/ml)410% (w/v) SDS7.5 μl(0.3%)5PI (100X)2.5 μl(1X)6H2O213.75 μl 7Total250 μl
0 Room temperature
ABC1ReagentAmountFinal Concentration210X NEBuffer 225 μl(1X)320 mg/ml BSA1.25 μl(0.1 mg/ml)410% (w/v) SDS7.5 μl(0.3%)5PI (100X)2.5 μl(1X)6H2O213.75 μl 7Total250 μl
No need to add BSA if the buffer already contains BSA, e.g., NEBuffer 2.1; Make fresh every time; Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer 2]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20 mg/ml BSA]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10% (w/v) SDS]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[PI (100X)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and pipet mix.
[PB2]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate/shake the sample tube in a thermal mixer for  at , .
37 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube.
[20% TritonX-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate/shake the sample tube in a thermal mixer for  at , .
37 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  (3%) aliquot in a new 1.5 ml microtube as a pre-digest DNA control (ctr-1), add  and store at  until the end of DAY2.  ABC1ReagentAmountFinal Concentration25 M NaCl250 μl(250 mM)30.5 M EDTA50 μl(5 mM)410% (w/v) SDS500 μl(1%)5H2O4.2 ml 6Total5 ml
8 µl
[DNA-preparation buffer]
-20 °C
ABC1ReagentAmountFinal Concentration25 M NaCl250 μl(250 mM)30.5 M EDTA50 μl(5 mM)410% (w/v) SDS500 μl(1%)5H2O4.2 ml 6Total5 ml
Store DNA-preparation buffer at  ; Warm the buffer at   in case the SDS precipitates during storage.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[5 M NaCl]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  .
[0.5 M EDTA]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  .
[10% (w/v) SDS]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-200 pipet.
200 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 or a P-200 pipet.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the washing buffer (WB) with NEBuffer DpnII (for DpnII digestion) or NEBuffer 2 (for HindIII digestion).  ABC1ReagentAmountFinal Concentration210X NEBuffer50 μl(1X)320 mg/ml BSA1.25 μl(0.1 mg/ml)420% (w/v) TritonX-1001.25 μl(0.05%)5H2O447.5 μl 6Total500 μl
ABC1ReagentAmountFinal Concentration210X NEBuffer50 μl(1X)320 mg/ml BSA1.25 μl(0.1 mg/ml)420% (w/v) TritonX-1001.25 μl(0.05%)5H2O447.5 μl 6Total500 μl
No need to add BSA if the buffer already contains BSA, e.g., NEBuffer 2.1; Do not use NEBuffer 3.1 for DpnII because non-specific cleavage (star activity) may be induced; Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20 mg/ml BSA]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20% (w/v) TritonX-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the restriction-enzyme mix on ice.  ABC1ReagentAmountFinal Concentration210X NEBuffer20 μl(1X)320 mg/ml BSA1 μl(0.1 mg/ml)4RE (50 U/ul)8 μl(400 U)520% Triton X-1000.5 μl(0.05%)6H2O170.5 μl 7Total200 μl
ABC1ReagentAmountFinal Concentration210X NEBuffer20 μl(1X)320 mg/ml BSA1 μl(0.1 mg/ml)4RE (50 U/ul)8 μl(400 U)520% Triton X-1000.5 μl(0.05%)6H2O170.5 μl 7Total200 μl
No need to add BSA if the buffer already contains BSA, e.g., NEBuffer 2.1; Do not use NEBuffer 3.1 for DpnII because non-specific cleavage (star activity) may be induced; For restriction enzymes at different concentration, e.g., HindIII at 100U/μl, adjust the volume with H2O; The restriction-enzyme mix may or may not contain Triton X-100; Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20 mg/ml BSA]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[RE (50 U/ul)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20% Triton X-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and pipet mix.
[restriction-enzyme mix]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate the sample tube in a thermal mixer for ~ at , .
37 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Take  (3%) aliquot in a new 1.5 ml microtube as a digested-DNA control (ctr-2), add  and store at  until the end of DAY 2.
This section includes the following stepsDNA fill-in reaction - Steps 85 - 106Ligation reaction - Steps 107 - 109
Follow cells/tissue resuspension steps as written, i.e., vortex mix or pipet mix.
6 µl
[DNA-preparation buffer]
-20 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-200 pipet.
100 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-200 pipet or a P-100 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare the washing buffer (WB).  ABC1ReagentAmountConcentration210X NEBuffer250 μl(1X)320 mg/ml BSA1.25 μl(0.1 mg/ml)420% (w/v) TritonX-1001.25 μl(0.05%)5H2O447.5 μl 6Total500 μl
ABC1ReagentAmountConcentration210X NEBuffer250 μl(1X)320 mg/ml BSA1.25 μl(0.1 mg/ml)420% (w/v) TritonX-1001.25 μl(0.05%)5H2O447.5 μl 6Total500 μl
No need to add BSA if the buffer already contains BSA, e.g., NEBuffer 2.1; Prepare enough buffer to perform four wash cycles or two wash cycles, for the DpnII-digested sample or the HindIII-digested sample respectively.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[10X NEBuffer2]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20 mg/ml BSA]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% (w/v) TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[H2O]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Repeat Steps 91 - 95 twice more for the DpnII sample (total of three wash cycles).
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare the DNA fill-in mix on ice.  ABC1ReagentAmountFinal Concentration210X NEBuffer 210 μl(1X)31 mM dCTP1.5 μl(15 μM)41 mM dGTP1.5 μl(15 μM)51 mM dTTP1.5 μl(15 μM)60.4 mM biotin-14-dATP3.75 μl(15 μM)7Klenow DNA polymerase (5 U/μl)6 μl(30 U)820% TritonX-1000.25 μl(0.05%)9H2O75.5 μl 10Total100 μl   ABC1ReagentAmountFinal Concentration210X NEBuffer 210 μl(1X)31 mM dATP1.5 μl(15 μM)41 mM dGTP1.5 μl(15 μM)51 mM dTTP1.5 μl(15 μM)60.4 mM biotin-14-dCTP3.75 μl(15 μM)7Klenow DNA polymerase (5 U/μl)3 μl(15 U)820% TritonX-1000.25 μl(0.05%)9H2O78.5 μl 10Total100 μl
ABC1ReagentAmountFinal Concentration210X NEBuffer 210 μl(1X)31 mM dCTP1.5 μl(15 μM)41 mM dGTP1.5 μl(15 μM)51 mM dTTP1.5 μl(15 μM)60.4 mM biotin-14-dATP3.75 μl(15 μM)7Klenow DNA polymerase (5 U/μl)6 μl(30 U)820% TritonX-1000.25 μl(0.05%)9H2O75.5 μl 10Total100 μl
ABC1ReagentAmountFinal Concentration210X NEBuffer 210 μl(1X)31 mM dATP1.5 μl(15 μM)41 mM dGTP1.5 μl(15 μM)51 mM dTTP1.5 μl(15 μM)60.4 mM biotin-14-dCTP3.75 μl(15 μM)7Klenow DNA polymerase (5 U/μl)3 μl(15 U)820% TritonX-1000.25 μl(0.05%)9H2O78.5 μl 10Total100 μl
The DNA fill-in mix may or may not contain Triton X-100.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[10X NEBuffer 2]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dCTP]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dGTP]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dTTP]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[0.4 mM biotin-14-dATP]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  for DNA fill-in mix for the DpnII-digested sample. Add  for DNA fill-in mix for the HindIII-digested sample.
[Klenow DNA polymerase (5 U/μl)]
[Klenow DNA polymerase (5 U/μl)]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  for DNA fill-in mix for the DpnII-digested sample. Add  for DNA fill-in mix for the HindIII-digested sample.
[H2O]
[H2O]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and pipet mix.
[DNA fill-in mix]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Incubate the sample tube in a thermal mixer for  at , .
25 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare the ligation mix on ice.  ABC1ReagentAmountFinal Concentration2T4 DNA ligase buffer (10X)10 μl(1X)3T4 DNA ligase (2,000 U/ul)2 μl(4,000 CEU)420% TritonX-1000.25 μl(0.05%)5H2O87.75 μl 6Total100 μl
ABC1ReagentAmountFinal Concentration2T4 DNA ligase buffer (10X)10 μl(1X)3T4 DNA ligase (2,000 U/ul)2 μl(4,000 CEU)420% TritonX-1000.25 μl(0.05%)5H2O87.75 μl 6Total100 μl
Prepare T4 DNA ligase buffer in small aliquots and store them at   when the buffer is thawed for the first time; Always use an aliquot that is not frozen and thawed repeatedly; The ligation mix may or may not contain Triton X-100; Use 2,000 CEU of T4 DNA ligase for the HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[T4 DNA ligase buffer (10X)]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[T4 DNA ligase (2,000 U/ul)]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[H2O]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and pipet mix.
[ligation mix]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Incubate the sample tube in a thermal mixer for - at , .
16 °C
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Prepare the DNA-extraction mix.  ABC1ReagentAmountFinal Concentration21M Tris-HCl (pH 8.0)2 μl(10 mM)30.5M EDTA2 μl(5 mM)410% SDS30 μl(1.5%)55M NaCl15 μl(375 mM)620 mg/ml Proteinase K10 μl(1 mg/ml)7H2O141 μl 8Total200 μl
ABC1ReagentAmountFinal Concentration21M Tris-HCl (pH 8.0)2 μl(10 mM)30.5M EDTA2 μl(5 mM)410% SDS30 μl(1.5%)55M NaCl15 μl(375 mM)620 mg/ml Proteinase K10 μl(1 mg/ml)7H2O141 μl 8Total200 μl
Prepare DNA-extraction mix at  .
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[1M Tris-HCl (pH 8.0)]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[0.5M EDTA]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[10% SDS]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[5M NaCl]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[20 mg/ml Proteinase K]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[H2O]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add  to the sample tube (from Step 106) and mix gently.
[DNA-extraction mix]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add  and  to the control tubes also (ctr-1 from Step 71 and ctr-2 from Step 85).
[DNA-extraction mix]
[H2O]
The total amount of the mixture will be  .
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Incubate the sample tubes in a thermal mixer for ~ at , .
65 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Take the sample tubes out from the thermal mixer (from Step 113) and let them cool down to .
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[RNase A (10 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Incubate sample tube in a thermal mixer for  at , .
37 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[Proteinase K (20 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Incubate the sample tube in a thermal mixer for  at , .
55 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Take out the sample tubes from the thermal mixer, let them cool down to , and proceed with DNA extraction.
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[Phenol/Chloroform/Isoamyl alcohol solution]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Transfer ~ of the aqueous phase into a new 1.5 ml microtube.
270 µl
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  ( pH 8.0, , ) to the sample tube containing Phenol and mix gently.
[TE/NaCl solution]
[Tris-HCl]
[EDTA]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Transfer ~ of the aqueous phase into the microtube containing the first aqueous phase.
300 µl
The total volume of the collected aqueous phase will be ~ .
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the collected aqueous phase and mix gently.
[glycogen solution (20 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the collected aqueous phase and mix gently until the solution becomes homogeneous.
[2-propanol]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Decant the supernatant, add , and mix gently to rinse the DNA pellet.
[70% EtOH]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Decant the supernatant.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Remove the supernatant completely with a P-100 or P-200 pipet, and keep the lid open for ~ to allow the ethanol to evaporate.
Do not over dry the pellet.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Dissolve the Hi-C DNA in  and the control DNAs (ctr-1 and ctr-2) in .
[EB]
[EB]
Mix gently to avoid shearing of the DNA; Store DNA samples at   or  .
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Quantitate DNA using  of the Hi-C DNA sample with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 7. (DAY 3) Hi-C DNA QC (QC1)]
Take - of the DNA sample (ctr-1, ctr-2, and Hi-C DNA) from Step 134 in a new PCR tube and adjust the concentration to 2-20 ng/μl with EB.
1 µl
2 µl
The concentration of the DNA samples in a trio (ctr-1, ctr-2, and Hi-C DNA) should be within two-fold of difference.
?. [Section 7. (DAY 3) Hi-C DNA QC (QC1)]
Analyze  of the DpnII-digested DNA samples (ctr-2 and Hi-C DNA) using the Agilent Bioanalyzer with the DNA High Sensitivity chip, or analyze  of the HindIII-digested DNA samples (ctr-1, ctr-2, and Hi-C DNA) using the Agilent TapeStation with the genomic tape.
1 µl
1 µl
Ctr-1 (pre-digested DNA) is >50 kb in size and cannot be analyzed using the Agilent Bioanalyzer with the DNA High Sensitivity chip.
?. [Section 7. (DAY 3) Hi-C DNA QC (QC1)]
Check the pattern of size shift between samples and the level of DNA degradation.
Only the qualified Hi-C DNA, showing the expected pattern of size shift with no or minimum degree of DNA degradation, is used for the preparation of the Hi-C library.
?. [Section 7. (DAY 3) Hi-C DNA QC (QC1)]
Store Hi-C DNA at  or proceed to the next step.
-20 °C
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Take  -  of Hi-C DNA (from the previous step) in a 0.2 ml PCR tube and adjust the total volume to  with H2O.
250 ng
2 µg
30 µl
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Prepare the T4-DNA-polymerase mix on ice.  ABC1ReagentAmountFinal Concentration210X NEBuffer 25 μl(1X)320 mg/ml BSA0.5 μl(0.2 mg/ml)410 mM GTP0.5 μl(100 μM)5T4 DNA pol (3 U/ul)1.67 μl(5 U)6H2O12.33 μl 7Total20 μl
DNA fill-in mix for the HindIII-digested sample is shown in the next step.
ABC1ReagentAmountFinal Concentration210X NEBuffer 25 μl(1X)320 mg/ml BSA0.5 μl(0.2 mg/ml)410 mM GTP0.5 μl(100 μM)5T4 DNA pol (3 U/ul)1.67 μl(5 U)6H2O12.33 μl 7Total20 μl
The DNA fill-in mix may or may not contain Triton X-100.
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[10X NEBuffer 2]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[20 mg/ml BSA]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[10 mM GTP]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[T4 DNA pol (3 U/ul)]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[H2O]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Prepare the T4-DNA-polymerase mix on ice.  ABC1ReagentAmountFinal Concentration210X NEBuffer 25 μl(1X)320 mg/ml BSA0.5 μl(0.2 mg/ml)410 mM ATP0.5 μl(100 μM)510 mM GTP0.5 μl(100 μM)6T4 DNA pol (3 U/ul)0.84 μl(2.5 U)7H2O12.66 μl 8Total20 μl
ABC1ReagentAmountFinal Concentration210X NEBuffer 25 μl(1X)320 mg/ml BSA0.5 μl(0.2 mg/ml)410 mM ATP0.5 μl(100 μM)510 mM GTP0.5 μl(100 μM)6T4 DNA pol (3 U/ul)0.84 μl(2.5 U)7H2O12.66 μl 8Total20 μl
The DNA fill-in mix may or may not contain Triton X-100.
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[10X NEBuffer 2]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[20 mg/ml BSA]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[10 mM ATP]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[10 mM GTP]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[T4 DNA pol (3 U/ul)]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add .
[H2O]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Add  to the Hi-C DNA and mix gently.
[T4-DNA-polymerase mix]
?. [Section 8. (DAY 3) Removal of biotin from un-ligated DNA ends]
Incubate the sample in a PCR machine for  at , and  at .
37 °C
75 °C
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Turn on the Covaris (S220 or E220).
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Transfer the entire reaction from Step 144 into a Covaris microTUBE and add .
[TE buffer]
The total volume will be  .
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Perform sonication Duty factor: 5%Peak incident power: 175Cycles per burst: 200Time:  ×2 Temperature:
7 °C
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Transfer  of the fragmented DNA into a 1.5 ml microtube.
120 µl
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Add  (×0.6 amount) of AMPure XP beads to the fragmented DNA, vortex and incubate  at .
72 µl
0 Room temperature
Bead-bound DNA larger than 600 bp is removed in the following steps.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Transfer the supernatant into a new 1.5 ml microtube.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Add  (×0.9 amount) of AMPure XP beads to the supernatant, vortex mix, and wait  at .
108 µl
0 Room temperature
Bead-bound DNA larger than 150 bp is collected in the following steps.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Remove the supernatant using a P-200 pipet while the sample tube is still on the magnet.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Add  to the beads while the sample tube is still on the magnet and wait . (1/2)
[80% EtOH]
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Remove EtOH with P-200 pipet while the sample tube is still on the magnet. (1/2)
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Add  to the beads while the sample tube is still on the magnet and wait . (2/2)
[80% EtOH]
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Remove EtOH with P-200 pipet while the sample tube is still on the magnet. (2/2)
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Quick spin the sample tube, put on the magnet and remove the residual EtOH completely using a P-10 or a P-10 pipet.
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Air dry the beads for ~ at  with the lid kept open.
0 Room temperature
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Add  to the beads, vortex mix and incubate  at .
[EB]
0 Room temperature
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Quick spin the sample tube, put on the magnet and collect the eluate in a new PCR tube.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Prepare the 2X binding-and-washing buffer (BWB).  ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 7.5)400 μl(10 mM)30.5 M EDTA (pH 8.0)80 μl(1 mM)45 M NaCl16 ml(2 M)510% Tween2080 μl(0.02%)6H2O23.44 ml 7Total40 ml
ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 7.5)400 μl(10 mM)30.5 M EDTA (pH 8.0)80 μl(1 mM)45 M NaCl16 ml(2 M)510% Tween2080 μl(0.02%)6H2O23.44 ml 7Total40 ml
Store at  .
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add .
[1 M Tris-HCl (pH 7.5)]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add .
[0.5 M EDTA (pH 8.0)]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add .
[5 M NaCl]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add .
[10% Tween20]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add .
[H2O]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Prepare 1X BWB by diluting the 2X BWB with H2O.
Store at  .
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Mix the bottle of the streptavidin beads and transfer ( × n) + 5% extra volume of beads into a new 1.5 ml microtube.
25 µl
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Put the tube on the magnetic and wait until the supernatant becomes clear.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, take off the tube from the magnet, add  (prepared in the first step of this section), and vortex mix.
[1X BWB]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, take off the tube from the magnet, add ( × n) + 5% extra volume of 2X BWB, and pipet mix.
60 µl
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add  (in 2X BWB) to the size-selected Hi-C DNA (prepared in previous section) and vortex mix.
[beads]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Incubate the sample tube in a thermal mixer for  at  with periodical mixing for  at  every .
20 °C
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (1/4)
[1X BWB]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (1/4)
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (2/4)
[1X BWB]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (2/4)
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (3/4)
[1X BWB]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (3/4)
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (4/4)
[1X BWB]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (4/4)
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, add , and vortex mix.
[EB]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, add  with the sample tube kept on the magnet.
[EB]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant, take off the sample tube from the magnet, quick spin, and put the sample tube back on the magnet.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the residual EB with a P-10 or a P-20 pipet.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add  and store the sample tube at  until the next day.
[EB]
4 °C
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the end-repair mix on ice  AB1ReagentAmount210X KAPA End Repair Buffer1.4 μl3KAPA End Repair Enzyme Mix1.0 μl4H2O11.6 μl5Total volume14 μl
This section consists of the following steps: End repair - Steps 187 - 204A-tailing - Steps 205 - 222Adapter ligation - Steps 223 - 240Pre-PCR (releasing DNA off the streptavidin beads) - Steps 241 - 255PCR cycle pre-determination - Steps 256 - 260Amplification of the library for Hi-C library QC (QC2) - Steps 261 - 276Library amplification - Steps 277 - 292
Library preparation is performed using the KAPA LTP DNA library kit but in a 1/5 reaction volume of the original protocol and with an additional step for PCR cycle pre-determination.
AB1ReagentAmount210X KAPA End Repair Buffer1.4 μl3KAPA End Repair Enzyme Mix1.0 μl4H2O11.6 μl5Total volume14 μl
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10X KAPA End Repair Buffer]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[KAPA End Repair Enzyme Mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the tube (from the previous section), put on the magnetic and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, add  to the beads and pipet mix.
[end-repair mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Incubate for  at  (followed by a hold at ) in a PCR machine.
20 °C
4 °C
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (1/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (2/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (2/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (3/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (3/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (4/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (4/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, add , and vortex mix.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, add  with the sample tube kept on the magnet.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, quick spin, and put the sample tube back on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the residual EB with a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Close the lid and put on ice.
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the A-tailing mix on ice.  AB1ReagentAmount210X KAPA A-Tailing Buffer1.0 μl3KAPA A-Tailing Enzyme0.6 μl4H2O8.4 μl5Total volume10 μl
AB1ReagentAmount210X KAPA A-Tailing Buffer1.0 μl3KAPA A-Tailing Enzyme0.6 μl4H2O8.4 μl5Total volume10 μl
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10X KAPA A-Tailing Buffer]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[KAPA A-Tailing Enzyme]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads (from step 205) and pipet mix.
[A-tailing mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Incubate  at  (followed by a hold at ) in a PCR machine.
30 °C
4 °C
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (1/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (2/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (2/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (3/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (3/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (4/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (4/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, add , and vortex mix.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, add  with the sample tube kept on the magnet.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, quick spin, and put the sample tube back on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the residual EB with a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Close the lid and put on ice.
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the ligation-buffer mix (without the ligase enzyme) on ice.  AB1ReagentAmount25X KAPA Ligation Buffer2.0 μl3H2O6.0 μl4Total volume8 μl
AB1ReagentAmount25X KAPA Ligation Buffer2.0 μl3H2O6.0 μl4Total volume8 μl
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[ligation-buffer mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads (from Step 221), add , and pipet mix.
[ligation-buffer mix]
[1 μM Illumina TruSeq compatible adapter]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  and pipet mix.
[KAPA T4 DNA ligase]
?. [Section 11. (DAY 4) Hi-C library preparation]
Incubate for  at  (followed by a hold at ) in a PCR machine.
20 °C
4 °C
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (1/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (2/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (2/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (3/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (3/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, add , and vortex mix. (4/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear. (4/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, add , and vortex mix.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, add  with the sample tube kept on the magnet.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, quick spin, and put the sample tube back on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the residual EB with a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Close the lid and put on ice.
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the pre-PCR mix.  AB1ReagentAmount22X KAPA HiFi Ready Mix10 μl310 μM TPC mix0.9 μl4H2O9.1 μl5Total volume20 μl
AB1ReagentAmount22X KAPA HiFi Ready Mix10 μl310 μM TPC mix0.9 μl4H2O9.1 μl5Total volume20 μl
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[2X KAPA HiFi Ready Mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10 μM TPC mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads (from Step 239) and pipet mix.
[pre-PCR mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Perform 4 cycles of PCR amplification at 98°C 45 sec, 4 cycles of (98°C 15 sec, 60°C 30 sec, 72°C 30 sec), 72°C 1 min, and a hold at 4°C.
?. [Section 11. (DAY 4) Hi-C library preparation]
Put the sample tube on the magnetic and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Transfer the supernatant to a new PCR tube, add  (×1 volume) of AMPure XP beads, vortex mix, and wait  at .
20 µl
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant using a P-200 pipet while the sample tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads while the sample tube is still on the magnet and wait . (1/2)
[80% EtOH]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (1/2)
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads while the sample tube is still on the magnet and wait . (2/2)
[80% EtOH]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (2/2)
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnet and remove the residual EtOH completely using a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Air dry the beads for ~ at  with the lid kept open.
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads, vortex mix, and incubate  at .
[EB]
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnet and collect the eluate in a new PCR tube.
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the real-time PCR mix.  AB1ReagentAmount22X KAPA HiFi HS real-time Mix5 μl310 μM TPC mix0.35 μl4H2O3.15 μl5Total volume8.5 μl
AB1ReagentAmount22X KAPA HiFi HS real-time Mix5 μl310 μM TPC mix0.35 μl4H2O3.15 μl5Total volume8.5 μl
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[2X KAPA HiFi HS real-time Mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10 μM TPC mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Dispense  in a well of a PCR plate (384 well or 96 well) and add  of the ligated DNA (from Step 254).
[real-time-PCR mix]
1.5 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Dispense  of each Fluorescence Standards in the same plate in separate wells.
10 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Run real-time PCR in a ROX minus condition at, 98°C 45 sec,  20 cycles of (98°C 15 sec, 60°C 30 sec, 72°C 30 sec), 72°C 1 min.
?. [Section 11. (DAY 4) Hi-C library preparation]
Determine the threshold-PCR-cycle (Ct) reaching Fluorescence Standard 1 but not exceeding Fluorescence Standard 2.
DNA for the library QC (QC2) is prepared by amplifying a small aliquot ( ) of the pre-PCR product with (Ct +3) cycles of PCR. Hi-C library for sequencing is prepared by amplifying the remaining 8.5 μl of the pre-PCR product (from Step 251) with the pre-determined Ct cycle.
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the PCR-mix for Hi-C library QC (QC2).  AB1ReagentAmount22X KAPA HiFi HotStart Ready Mix5 μl310 μM TPC0.45 μl4H2O3.55 μl5Total volume9 μl
AB1ReagentAmount22X KAPA HiFi HotStart Ready Mix5 μl310 μM TPC0.45 μl4H2O3.55 μl5Total volume9 μl
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[2X KAPA HiFi HotStart Ready Mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10 µM TPC]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Dispense of the 'QC2-PCR mix' in a PCR tube, add  of the pre-PCR product (from Step 254).
9 µl
1 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Perform PCR amplification at, 98°C 45 sec, (Ct +3) cycles of (98°C 15 sec, 60°C 30 sec, 72°C 30 sec), 72°C 1 min, and a hold at 4°C.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  (×1 volume) of AMPure XP beads, vortex mix, and wait  at .
10 µl
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant using a P-200 pipet while the sample tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads while the sample tube is still on the magnet and wait . (1/2)
[80% EtOH]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (1/2)
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads while the sample tube is still on the magnet and wait . (2/2)
[80% EtOH]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (2/2)
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnet and remove the residual EtOH completely using a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Air dry the beads for ~ at  with the lid kept open.
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads, vortex mix, and incubate  at .
[EB]
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnet and collect the eluate in a new PCR tube.
?. [Section 11. (DAY 4) Hi-C library preparation]
Quantitate the DNA using  of the DNA sample with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Proceed to the Hi-C library QC (QC2).
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare a PCR mix for Hi-C library amplification.  AB1ReagentAmount22X KAPA HiFi HotStart Ready Mix10 μl310 μM TPC0.9 μl4H2O0.6 μl5Total volume11.5 μl
AB1ReagentAmount22X KAPA HiFi HotStart Ready Mix10 μl310 μM TPC0.9 μl4H2O0.6 μl5Total volume11.5 μl
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[2X KAPA HiFi HotStart Ready Mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10 µM TPC]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Dispense  of the 'Library-amplification-PCR mix' to the tube containing the pre-PCR product (from Step 254).
11.5 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Perform PCR amplification at, 98°C 45 sec,  (pre-determined Ct) cycles of (98°C 15 sec, 60°C 30 sec, 72°C 30 sec), 72°C 1 min, and a hold at 4°C.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  (×1 volume) of AMPure XP beads, vortex mix, and wait  at .
20 µl
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant using a P-200 pipet while the sample tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads while the sample tube is still on the magnet and wait . (1/2)
[80% EtOH]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (1/2)
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads while the sample tube is still on the magnet and wait . (2/2)
[80% EtOH]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (2/2)
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnet and remove the residual EtOH completely using a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Air dry the beads for ~ at  with the lid kept open.
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads, vortex mix, and incubate  at .
[EB]
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on the magnet and collect the eluate in a new microtube (1.5 ml).
?. [Section 11. (DAY 4) Hi-C library preparation]
Quantitate the DNA using  of the DNA sample with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Analyze the size-distribution using  of the library DNA with Agilent TapeStation High Sensitivity D1000 tape.
1 µl
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Prepare the restriction-mix for QC2 with or without the restriction enzyme.   AB1ReagentAmount210X buffer1 μl3RE (10 U/ul) or H2O0.3 μl420 mg/ml BSA0.05 μl5H2O&nbsp;μl6Total volumeµl
AB1ReagentAmount210X buffer1 μl3RE (10 U/ul) or H2O0.3 μl420 mg/ml BSA0.05 μl5H2O&nbsp;μl6Total volumeµl
Cut the DpnII-digested library with ClaI, and the HindIII-digested library with NheI.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Dispense 'QC2-restriction mix' (with or without the RE) to a PCR tube, add - of the amplified library DNA, adjust the total volume of the reaction to , and incubate  at .
10 ng
20 ng
10 µl
37 °C
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Add  (×1.8 volume) of AMPure XP beads, vortex mix, and wait  at .
18 µl
0 Room temperature
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Quick spin the sample tube, put on the magnetic and wait until the supernatant becomes clear.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Remove the supernatant using a P-200 pipet while the sample tube is still on the magnet.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Add  to the beads while the sample tube is still on the magnet and wait . (1/2)
[80% EtOH]
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (1/2)
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Add  to the beads while the sample tube is still on the magnet and wait . (2/2)
[80% EtOH]
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (2/2)
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Quick spin the sample tube, put on the magnet and remove the residual EtOH completely using a P-10 or a P-20 pipet.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Air dry the beads for ~ at  with the lid kept open.
0 Room temperature
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Add  to the beads, vortex mix, and incubate  at .
[EB]
0 Room temperature
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Quick spin the sample tube, put on the magnet and collect the eluate in a new PCR tube.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Analyze the size-shift of the library using  of the DNA with Agilent TapeStation High Sensitivity D1000 tape.
2 µl
Place RE(-) and RE(+) samples for the same library side-by-side for easy comparison.
?. [Section 2. (DAY 0) Fixation of cells]
Store the sample tubes in an ultra-low temperature freezer (e.g., ) until use.
-80 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  (3%) aliquot (from Step 39) in a new 1.5 ml microtube as a pre-digest DNA control (ctr-1), add  and store at  until the end of DAY2.
8 µl
[DNA-preparation buffer]
-20 °C
?. [Section 2. (DAY 0) Fixation of cells]
Repeat the washing cycle (Steps 16-19) once more.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  (3%) aliquot in a new 1.5 ml microtube as a digested-DNA control (ctr-2), add  and store at  until the end of DAY 2.
6 µl
[DNA-preparation buffer]
-20 °C
?. [Section 9. (DAY 3) Fragmentation and size selection of the Hi-C DNA]
Repeat the washing cycle (Steps 126-127) once more.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Repeat the washing cycle (Steps 145-148) three more times.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add  to the sample tube and vortex mix. (1/4)
[1X BWB]
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Remove the supernatant while the tube is still on the magnet.(1/4)
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Take off the sample tube from the magnet.
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Take off the sample tube from the magnet.(1/4)
?. [Section 10. (DAY 3) Enrichment of biotin-containing DNA]
Add  with the tube kept on the magnet.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant while the tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Take off the sample tube from the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the sample tube and vortex mix. (1/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant while the tube is still on the magnet.(1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear. (1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Take off the sample tube from the magnet.(1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Repeat the washing cycle (Steps 164-167) three more times.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  and vortex mix.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant while the tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  with the tube kept on the magnet.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, quick spin, and put the sample tube back on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the residual EB with a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the sample tube and vortex mix. (1/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant while the tube is still on the magnet.(1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear. (1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Take off the sample tube from the magnet.(1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Repeat the washing cycle (Steps 182-185) three more times.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  and vortex mix.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant while the tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  with the tube kept on the magnet.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, quick spin, and put the sample tube back on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the residual EB with a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Close the lid and put on ice.
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant while the tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Take off the sample tube from the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant while the tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Take off the sample tube from the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the sample tube and vortex mix. (1/4)
[1X BWB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant while the tube is still on the magnet.(1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear. (1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Take off the sample tube from the magnet.(1/4)
?. [Section 11. (DAY 4) Hi-C library preparation]
Repeat the washing cycle (Steps 201-204) three more times.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  and vortex mix.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant while the tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  with the tube kept on the magnet.
[EB]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant, take off the sample tube from the magnet, quick spin, and put the sample tube back on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the residual EB with a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Close the lid and put on ice.
?. [Section 11. (DAY 4) Hi-C library preparation]
Repeat the washing cycle (Steps 220-221) once more.
?. [Section 11. (DAY 4) Hi-C library preparation]
Prepare the PCR-mix for Hi-C library amplification.  AB1ReagentAmount22X KAPA HiFi HotStart Ready Mix10 μl310 μM TPC0.9 μl4H2O0.6 μl5Total volume11.5 μl
AB1ReagentAmount22X KAPA HiFi HotStart Ready Mix10 μl310 μM TPC0.9 μl4H2O0.6 μl5Total volume11.5 μl
Make 5% extra volume.
?. [Section 11. (DAY 4) Hi-C library preparation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[2X KAPA HiFi HotStart Ready Mix]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add .
[10 µM TPC]
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  of the PCR-mix for Hi-C library amplification to the tube containing the pre-PCR product (from Step 226).
11.5 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Perform PCR amplification at, 98°C 45 sec,  (Ct) cycles of (98°C 15 sec, 60°C 30 sec, 72°C 30 sec), 72°C 1 min, and a hold at 4°C.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  (×1 volume) of AMPure XP beads, vortex mix, and wait  at .
20 µl
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove the supernatant using a P-200 pipet while the sample tube is still on the magnet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads while the sample tube is still on the magnet and wait . (1/2)
[80% EtOH]
?. [Section 11. (DAY 4) Hi-C library preparation]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (1/2)
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and remove the residual EtOH completely using a P-10 or a P-20 pipet.
?. [Section 11. (DAY 4) Hi-C library preparation]
Air dry the beads for ~ at  with the lid kept open.
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Add  to the beads, vortex mix, and incubate  at .
[EB]
0 Room temperature
?. [Section 11. (DAY 4) Hi-C library preparation]
Quick spin the sample tube, put on magnet and collect the eluate in a new microtube (1.5 ml).
?. [Section 11. (DAY 4) Hi-C library preparation]
Quantitate the DNA using  of the DNA sample with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Analyze the size-distribution using  of the library DNA using the Agilent TapeStation with the High Sensitivity D1000 tape.
2 µl
?. [Section 11. (DAY 4) Hi-C library preparation]
Repeat the washing cycle (Steps 241-242) once more.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Add  (×1 volume) of AMPure XP beads to the PCR product from the previous section (Step 234), vortex mix, and wait  at .
10 µl
0 Room temperature
DNA larger than 200 bp will bind to the AMPure XP beads.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Quick spin the sample tube, put on magnet and wait until the supernatant becomes clear.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Remove the supernatant using a P-200 pipet while the sample tube is still on the magnet.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Add  to the beads while the sample tube is still on the magnet and wait . (1/2)
[80% EtOH]
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Remove EtOH with a P-200 pipet while the sample tube is still on the magnet. (1/2)
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Repeat the washing cycle (Steps 253-254) once more.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Quick spin the sample tube, put on magnet and remove the residual EtOH completely using a P-10 or a P-20 pipet.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Air dry the beads for ~ at  with the lid kept open.
0 Room temperature
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Add  to the beads, vortex mix, and incubate  at .
[EB]
0 Room temperature
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Quick spin the sample tube, put on magnet and collect the eluate in a new PCR tube.
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Quantitate the DNA using  of the DNA sample with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Prepare the restriction-mix for QC2 with or without the restriction enzyme (RE)  AB1ReagentAmount210X buffer1 μl3RE (10 U/μl) or H2O0.3 μl420 mg/ml BSA0.05 μl5H2Oμl6Total volumeµl
AB1ReagentAmount210X buffer1 μl3RE (10 U/μl) or H2O0.3 μl420 mg/ml BSA0.05 μl5H2Oμl6Total volumeµl
Adjust the volume of H2O based on the concentration of the amplified library DNA.The restriction enzyme for the DpnII-digested library is ClaI.The restriction enzyme for the HindIII-digested library is NheI.Make 5% extra volume.
?. [Section 2. (DAY 0) Fixation of the tissue]
Add 1/20 volume of a  to quench the formaldehyde.
[glycine solution]
?. [Section 2. (DAY 0) Fixation of the tissue]
Repeat the washing cycle (Steps 21-24) once more.
?. [Section 2. (DAY 0) Fixation of the tissue]
Store the sample tubes in an ultra-low temperature freezer (e.g., ) until use.
-80 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Take one microtube out from the ultra-low temperature freezer (from Step 29) and immediately add .
[lysis buffer]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Incubate the sample tube for ~ at , in a heat block.
65 °C
Use a heat block equipped with a heated lid to avoid condensation of water on the lid.Alternatively, the incubation can be performed in a heated oven.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Take the sample tube out from the heat block (from Step 33) and let it cool down to .
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Quantitate the DNA using  of the sample with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Calculate the total amount of DNA extracted from the tissue pellet used for DNA extraction.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Add 1/20 volume of a  to quench the formaldehyde.
[glycine solution]
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Repeat the washing cycle (Steps 21-24) once more.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Store the sample tubes in an ultra-low temperature freezer (e.g., ) until use.
-80 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Prepare lysis buffer.  ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl300 μl(300 mM)40.5 M EDTA (pH 8.0)50 μl(5 mM)520% (w/v) SDS250 μl(1%)6H2O4.35 ml7Total5 ml
This section includes the following stepsCell/tissue lysis - Steps 30 - 38DNA extraction and quantification - Steps 39 - 57
The pre-determination section quantitates the amount of DNA contained in a cell/tissue pellet prepared in the previous section. Pre-determination should always be performed for tissue samples, but it is optional when the DNA content in a pellet is estimable,. e.g., for cultured cells and nucleated red blood cells.
Follow cells/tissue resuspension steps as written, i.e., gently mix or pipet mix.
ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl300 μl(300 mM)40.5 M EDTA (pH 8.0)50 μl(5 mM)520% (w/v) SDS250 μl(1%)6H2O4.35 ml7Total5 ml
Store lysis buffer at  .Warm the lysis buffer at   to dissolve precipitates formed during storage.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Take one microtube out from the ultra-low temperature freezer (from Step 30) and immediately add .
[lysis buffer]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and pipet mix.
[Proteinase K (20 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Incubate the sample tube for ~ at , in a heat block.
65 °C
Use a heat block equipped with a heated lid to avoid condensation of water on the lid inside the tube.Alternatively, the incubation can be performed in a heated oven.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Take the sample tube out from the heat block (from Step 33) and let it cool down to .
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and mix gently.
[RNase A (10 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Incubate the sample tube in a thermal mixer for at , .
37 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and mix gently.
[Proteinase K (20 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Incubate the sample tube in a thermal mixer for  at , .
55 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Take the sample tube out from the thermal mixer, let it cool down to , and proceed with DNA extraction.
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and mix gently.
[Phenol/Chloroform/Isoamyl alcohol solution]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Transfer ~ of the aqueous phase into a new 1.5 ml microtube.
270 µl
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  ( pH 8.0, , ) to the sample tube containing Phenol and mix gently.
[TE/NaCl solution]
[Tris-HCl]
[NaCl]
[EDTA]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Transfer ~of the aqueous phase into the microtube containing the first aqueous phase.
300 µl
The total volume of the collected aqueous phase will be ~ .
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the collected aqueous phase and mix gently.
[glycogen solution (20 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add to the collected aqueous phase and mix gently until the solution becomes homogeneous.
[2-Propanol]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Decant the supernatant, add , and mix gently to rinse the DNA pellet.
[70% EtOH]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Decant the supernatant.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Remove the supernatant completely with a P-100 or a P-200 pipet, and keep the lid open for ~ to allow the ethanol to evaporate.
Do not over dry the pellet.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Dissolve the DNA in -.
30 µl
[TE]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Quantitate the DNA using  of the sample with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Calculate the total amount of DNA extracted from the tissue pellet used for DNA extraction.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Determine the number of tissue pellets to use for Hi-C.
A tissue pellet that contains  -  of DNA is used for Hi-C.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Prepare TE/NaCl solution.  ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl250 μl(250 mM)40.5 M EDTA (pH 8.0)10 μl(1 mM)5H2O4.69 ml6Total5 ml
ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl250 μl(250 mM)40.5 M EDTA (pH 8.0)10 μl(1 mM)5H2O4.69 ml6Total5 ml
Store TE/NaCl solution at  .
?. [Section 12. (DAY 4) Quality control of the Hi-C library (QC2)]
Repeat the washing cycle (Steps 266-267) once more.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the permeabilization buffer 1 (PB1).  ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 8.0)400 μl(10 mM)35 M NaCl80 μl(10 mM)410% (w/v) NP-40800 μl(0.2%)5H2O38.72 ml 6Total40 ml
This section includes the following steps.Cell/tissue permeabilization - Steps 58 - 83Restriction enzyme digestion - Steps 84 - 86
The number of cells to use for Hi-C is determined based on the amount of DNA.Use 1×106  cells for an animal with the genome size of 3-3.5 Gb (e.g., human and mouse), or 2×106 cells for an animal with the genome size of 1-1.5 Gb (e.g., chicken and western clawed frog).For tissue samples, use a pellet that contains 2-10 μg of DNA.
Follow cells/tissue resuspension steps as written, i.e., vortex mix or pipet mix.
ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 8.0)400 μl(10 mM)35 M NaCl80 μl(10 mM)410% (w/v) NP-40800 μl(0.2%)5H2O38.72 ml 6Total40 ml
Filtrate and store at  .
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take ( ×n) + 5% extra volume of PB1 in a new tube, add 1/100 vol of a proteinase inhibitor cocktail (PI), and put on ice.
400 µl
Add PI to PB1 just before use.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take the frozen tissue pellet out from the freezer (from Step 29) and immediately add .
[PB1 (with PI)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Vortex mix.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate the sample tube on ice for  with periodical mixing every -.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-1000 pipet.
300 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 or a P-200 pipet, and put the sample tube on ice.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the permeabilization buffer 2 (PB2) at .  ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 2.125 μl(1X)310% (w/v) SDS7.5 μl(0.3%)4PI (100X)2.5 μl(1X)5H2O215 μl 6Total250 μl
0 Room temperature
ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 2.125 μl(1X)310% (w/v) SDS7.5 μl(0.3%)4PI (100X)2.5 μl(1X)5H2O215 μl 6Total250 μl
Make fresh every time.Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  in a tube of appropriate size.
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer 2.1]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10% (w/v) SDS]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[PI (100X)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and pipet mix.
[PB2]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate/shake the sample tube in a thermal mixer for  at , .
37 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube.
[20% TritonX-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate/shake the sample tube in a thermal mixer for  at , .
37 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the DNA-preparation buffer.   ABC1ReagentAmountFinal Concentration25 M NaCl250 μl(250 mM)30.5 M EDTA50 μl(5 mM)410% (w/v) SDS500 μl(1%)5H2O4.2 ml 6Total5 ml
ABC1ReagentAmountFinal Concentration25 M NaCl250 μl(250 mM)30.5 M EDTA50 μl(5 mM)410% (w/v) SDS500 μl(1%)5H2O4.2 ml 6Total5 ml
Store DNA-preparation buffer at  .Warm the DNA-preparation buffer at   to dissolve precipitates formed during storage.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  (3%) aliquot (from Step 71) in a new 1.5 ml microtube as a pre-digest DNA control (ctr-1), add  and store at  until the end of DAY2.
8 µl
[DNA-preparation buffer]
-20 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-200 pipet.
200 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 or a P-200 pipet, and put the sample tube on ice.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare washing buffer (WB) with NEBuffer DpnII (for the DpnII digested sample).  ABC1ReagentAmount (x1 wash cycle)Final Concentration210X NEBuffer DpnII50 μl(1X)320 mg/ml BSA2.5 μl(0.1 mg/ml)420% (w/v) TritonX-1001.25 μl(0.05%)5H2O446.25 μl 6Total500 μl
ABC1ReagentAmount (x1 wash cycle)Final Concentration210X NEBuffer DpnII50 μl(1X)320 mg/ml BSA2.5 μl(0.1 mg/ml)420% (w/v) TritonX-1001.25 μl(0.05%)5H2O446.25 μl 6Total500 μl
Do not use NEBuffer 3.1 for the samples for DpnII digestion.Make WB for HindIII digestion with NEBuffer 2.1 (that contains BSA).Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  in a tube of appropriate size.
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer DpnII]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20 mg/ml BSA]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20% (w/v) TritonX-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet, and put the sample tube on ice.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the restriction-enzyme mix (for DpnII digestion) on ice.  ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer DpnII20 μl(1X)320 mg/ml BSA1 μl(0.1 mg/ml)4DpnII (50 U/μl)8 μl(400 U)520% Triton X-1000.5 μl(0.05%)6H2O170.5 μl 7Total200 μl
ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer DpnII20 μl(1X)320 mg/ml BSA1 μl(0.1 mg/ml)4DpnII (50 U/μl)8 μl(400 U)520% Triton X-1000.5 μl(0.05%)6H2O170.5 μl 7Total200 μl
Do not use NEBuffer 3.1 for DpnII, because non-specific cleavage (star activity) may be induced.Use NEBuffer 2.1 (that contains BSA) for HindIII digestion.For restriction enzymes at different concentration, e.g., HindIII at 100U/μl, adjust the volume with H2O.Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  in a tube of appropriate size.
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer DpnII]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20 mg/ml BSA]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[DpnII (50 U/μl)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20% Triton X-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and pipet mix.
[restriction-enzyme mix]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate the sample tube in a thermal mixer for ~ at , .
37 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  (3%) aliquot in a new 1.5 ml microtube as a digested-DNA control (ctr-2), add  and store at  until the end of DAY 2.
6 µl
[DNA-preparation buffer]
-20 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
This section includes the following steps.DNA fill-in reaction - Steps 88 - 110Ligation reaction - Steps 111 - 113
Follow cells/tissue resuspension steps as written, i.e., vortex mix or pipet mix.
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-200 pipet.
100 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-200 pipet or a P-100 pipet, and put the sample tube on ice.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare the washing buffer (WB) with NEBuffer 2.1.  ABC1ReagentAmount (x1 wash cycle)Concentration210X NEBuffer 2.150 μl(1X)320% (w/v) TritonX-1001.25 μl(0.05%)4H2O448.75 μl 5Total500 μl
ABC1ReagentAmount (x1 wash cycle)Concentration210X NEBuffer 2.150 μl(1X)320% (w/v) TritonX-1001.25 μl(0.05%)4H2O448.75 μl 5Total500 μl
Prepare buffer to perform total of four or two wash cycles (at Steps 93, 98, 105) for the DpnII-digested sample or the HindIII-digested sample respectively.Make 5% extra volume.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[10X NEBuffer 2.1]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% (w/v) TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Repeat the washing cycle (Steps 93-97) twice more for the DpnII-digested sample (total of three wash cycles).
Additional washing cycle is not required for the HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare the DNA fill-in mix on ice.  ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 210 μl(1X)30.4 mM biotin-14-dATP3.75 μl(15 μM)41 mM dCTP1.5 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)6 μl(30 U)820% TritonX-1000.25 μl(0.05%)9H2O75.5 μl 10Total100 μl   ABC1ReagentAmountFinal Concentration210X NEBuffer 210 μl(1X)31 mM dATP1.5 μl(15 μM)40.4 mM biotin-14-dCTP3.75 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)3 μl(15 U)820% TritonX-1000.25 μl(0.05%)9H2O78.5 μl 10Total100 μl
ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 210 μl(1X)30.4 mM biotin-14-dATP3.75 μl(15 μM)41 mM dCTP1.5 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)6 μl(30 U)820% TritonX-1000.25 μl(0.05%)9H2O75.5 μl 10Total100 μl
ABC1ReagentAmountFinal Concentration210X NEBuffer 210 μl(1X)31 mM dATP1.5 μl(15 μM)40.4 mM biotin-14-dCTP3.75 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)3 μl(15 U)820% TritonX-1000.25 μl(0.05%)9H2O78.5 μl 10Total100 μl
Make a DNA fill-in mix that contains either biotin-14-dATP or biotin-14-dCTP, for the DpnII-digested sample or the HindIII-digested sample respectively.Make 5% extra volume.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Take  in a tube of appropriate size.
[H2O]
Take   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[10X NEBuffer 2]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[0.4 mM biotin-14-dATP]
Add   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dCTP]
Add   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dGTP]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dTTP]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[Klenow DNA polymerase (5 U/μl)]
Add   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and pipet mix.
[DNA fill-in mix]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Incubate the sample tube in a thermal mixer for  at , .
25 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare ligation mix on ice.  ABC1ReagentAmountFinal Concentration2T4 DNA ligase buffer (10X)10 μl(1X)3T4 DNA ligase (2,000 U/ul)2 μl(4,000 CEU)420% TritonX-1000.25 μl(0.05%)5H2O87.75 μl 6Total100 μl
ABC1ReagentAmountFinal Concentration2T4 DNA ligase buffer (10X)10 μl(1X)3T4 DNA ligase (2,000 U/ul)2 μl(4,000 CEU)420% TritonX-1000.25 μl(0.05%)5H2O87.75 μl 6Total100 μl
Prepare T4 DNA ligase buffer in small aliquots and store them at   when the buffer is thawed for the first time.Always use an aliquot that is not thawed and frozen repeatedly.Use 2,000 CEU of T4 DNA ligase for the HindIII-digested sample.Make 5% extra volume.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[T4 DNA ligase buffer (10X)]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[T4 DNA ligase (2,000 U/ul)]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and pipet mix.
[ligation mix]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Incubate the sample tube in a thermal mixer for - at , .
16 °C
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Prepare DNA-extraction mix.  ABC1ReagentAmount (x1 reaction)Final Concentration21M Tris-HCl (pH 8.0)2 μl(10 mM)30.5M EDTA2 μl(5 mM)410% SDS30 μl(1.5%)55M NaCl15 μl(375 mM)620 mg/ml Proteinase K10 μl(1 mg/ml)7H2O141 μl 8Total200 μl
ABC1ReagentAmount (x1 reaction)Final Concentration21M Tris-HCl (pH 8.0)2 μl(10 mM)30.5M EDTA2 μl(5 mM)410% SDS30 μl(1.5%)55M NaCl15 μl(375 mM)620 mg/ml Proteinase K10 μl(1 mg/ml)7H2O141 μl 8Total200 μl
Prepare DNA-extraction mix at  .Make 5% extra volume.
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Take  in a tube of appropriate size.
[H2O]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[1M Tris-HCl (pH 8.0)]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[0.5M EDTA]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[10% SDS]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[5M NaCl]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[20 mg/ml Proteinase K]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add  to the sample tube (from Step 113) and mix gently.
[DNA-extraction mix]
The total amount of the mixture will be  .
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add  and  to the control tubes (ctr-1 from Step 73 and ctr-2 from Step 87) and mix gently.
[DNA-extraction mix]
[H2O]
The total amount of the mixture will be  .
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Incubate the sample tubes in a thermal mixer for ~ at , .
65 °C
Use a thermal mixer equipped with a heated lid to avoid condensation of water on the lid.Alternatively, the incubation can be performed in a heated oven.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Take the sample tube out from the thermal mixer (from Step 117) and let it cool down to .
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[RNase A (10 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Incubate the sample tube in a thermal mixer for  at , .
37 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[Proteinase K (20 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Incubate the sample tube in a thermal mixer for  at , .
55 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Take the sample tube out from the thermal mixer, let it cool down to , and proceed with DNA extraction.
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[Phenol/Chloroform/Isoamyl alcohol solution]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Transfer ~ of the aqueous phase into a new 1.5 ml microtube.
250 µl
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Prepare TE/NaCl solution.  ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl250 μl(250 mM)40.5 M EDTA (pH 8.0)10 μl(1 mM)5H2O4.69 ml6Total5 ml
ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl250 μl(250 mM)40.5 M EDTA (pH 8.0)10 μl(1 mM)5H2O4.69 ml6Total5 ml
Store TE/NaCl solution at  .
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube containing Phenol and mix gently.
[TE/NaCl solution]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Transfer ~ of the aqueous phase into the microtube containing the first aqueous phase.
300 µl
The total volume of the collected aqueous phase will be ~ .
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the collected aqueous phase and mix gently.
[glycogen solution (20 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the collected aqueous phase and mix gently until the solution becomes homogeneous.
[2-propanol]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Decant the supernatant, add , and mix gently to rinse the DNA pellet.
[70% EtOH]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Decant the supernatant.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Remove the supernatant completely with a P-100 or a P-200 pipet, and keep the lid open for ~ to allow the ethanol to evaporate.
Do not over dry the pellet.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Quantitate DNA using  of the DNA samples with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
The following procedure should be performed as described in the step-case for the dissociated cells.Start from Section 7. (DAY3) Hi-C DNA QC (QC1), Step 109.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Dissect and collect tissue in a microtube (1.5 or 2.0 ml) and freeze immediately in liquid nitrogen.
Store the frozen tissue in an ultra-low temperature freezer (e.g.,  ).
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Pre-cool the Tokken stainless-steel tubes and bullets in liquid nitrogen.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Transfer tissue (up to 4 mm3 in size) into each stainless-steel tube.
Break a large tissue into small pieces if necessary.Do not let the tissue thaw during the powderization process.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Put a bullet in the stainless-steel tube and close the screw cap.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Assemble the Tokken tube holder with three stainless-steel tubes.
The tube holder should always be assembled with three stainless-steel tubes to balance the weight.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Submerge the tube holder (with tubes) in liquid nitrogen and wait ~.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Take out the tube holder (with tubes) from liquid nitrogen and immediately place inside the Tokken acryl-grinder, then vigorously shake the grinder for about 100 strokes to powderize the tissue.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Remove the bullet and collect the powderized tissue in a microtube (1.5 or 2.0 ml) pre-cooled in liquid nitrogen.
Hand warm the lid of the deep-frozen microtube before closing its lid; otherwise the tube may break.
?. [Section 1. (DAY 0) Preparation of cells/tissue]
Proceed immediately to cell fixation (Step  11) or store the powderized tissue in an ultra-low temperature freezer (e.g., ) until use.
-80 °C
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Prepare the fixing solution and put on ice.  ABC1ReagentAmountFinal Concentration216% formaldehyde solution1 ml(1%)3PBS (-)15 ml　4total16 ml
ABC1ReagentAmountFinal Concentration216% formaldehyde solution1 ml(1%)3PBS (-)15 ml　4total16 ml
Make fresh every time.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Take a sample tube (microtube) out from the ultra-low temperature freezer, immediately add -.
1 ml
[ice-cold fixing solution]
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Vortex mix until the tissue powder is fully resuspended in the fixing solution.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Incubate the tissue-suspension for - in a heat block or a water bath set at .
25 °C
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Quick spin the sample tube.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Add 1/20 volume of a  to quench the formaldehyde.
[glycine solution]
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Vortex mix.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Put the sample tube on on ice for ~ .
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Centrifuge the sample tube. Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Remove the supernatant.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Add .  (1/2)
[ice-cold PBS (-)]
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Vortex mix. (1/2)
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Centrifuge the sample tube. (1/2) Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Remove the supernatant. (1/2)
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Repeat the washing cycle (Steps 21-24) once more.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Resuspend the pellet in - and aliquot the tissue-suspension evenly into five to ten microtubes (1.5 ml).
1 ml
[ice-cold PBS]
Aliquot a 4 mm3-size tissue into five to ten microtubes.Adjust the number of microtubes proportionally for a tissue at different size.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Centrifuge the sample tubes.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Remove the supernatant.
?. [Section 2. (DAY 0) Fixation of cells/tissue]
Store the sample tubes in an ultra-low temperature freezer (e.g., ) until use.
-80 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Prepare lysis buffer.  ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl300 μl(300 mM)40.5 M EDTA (pH 8.0)50 μl(5 mM)520% (w/v) SDS250 μl(1%)6H2O4.35 ml7Total5 ml
This section includes the following steps.Cell/tissue lysis - Steps 30 - 38DNA extraction and quantification - Steps 39 - 57
The pre-determination section quantitates the amount of DNA contained in a cell/tissue pellet prepared in the previous section. Pre-determination should always be performed for tissue samples, but it is optional when the DNA content in a pellet is estimable,. e.g., for cultured cells and nucleated red blood cells.
Follow cells/tissue resuspension steps as written, i.e., gently mix or pipet mix.
ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl300 μl(300 mM)40.5 M EDTA (pH 8.0)50 μl(5 mM)520% (w/v) SDS250 μl(1%)6H2O4.35 ml7Total5 ml
Store lysis buffer at  .Warm the lysis buffer at   to dissolve precipitates formed during storage.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Take one microtube out from the ultra-low temperature freezer (from Step 29) and immediately add .
[lysis buffer]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and pipet mix.
[Proteinase K (20 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Incubate the sample tube for ~ at , in a heat block.
65 °C
Use a heat block equipped with a heated lid to avoid condensation of water on the lid.Alternatively, the incubation can be performed in a heated oven.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Take the sample tube out from the heat block (from Step 33) and let it cool down to .
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and mix gently.
[RNase A (10 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Incubate the sample tube in a thermal mixer for at , .
37 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and mix gently.
[Proteinase K (20 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Incubate the sample tube in a thermal mixer for  at , .
55 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Take the sample tube out from the thermal mixer, let it cool down to , and proceed with DNA extraction.
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the sample tube and mix gently.
[Phenol/Chloroform/Isoamyl alcohol solution]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Transfer ~ of the aqueous phase into a new 1.5 ml microtube.
250 µl
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  ( pH 8.0, , ) to the sample tube containing Phenol and mix gently.
[TE/NaCl solution]
[Tris-HCl]
[NaCl]
[EDTA]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Transfer ~of the aqueous phase into the microtube containing the first aqueous phase.
300 µl
The total volume of the collected aqueous phase will be ~ .
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add  to the collected aqueous phase and mix gently.
[glycogen solution (20 mg/ml)]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Add to the collected aqueous phase and mix gently until the solution becomes homogeneous.
[2-Propanol]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Decant the supernatant, add , and mix gently to rinse the DNA pellet.
[70% EtOH]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Decant the supernatant.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Remove the supernatant completely with a P-100 or a P-200 pipet, and keep the lid open for ~ to allow the ethanol to evaporate.
Do not over dry the pellet.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Dissolve the DNA in -.
30 µl
[TE]
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Quantitate the DNA using  of the sample with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Calculate the total amount of DNA extracted from the tissue pellet used for DNA extraction.
?. [Section 3. (DAY 0) Pre-determination of the amount of tissue to use for Hi-C]
Determine the amount of fixed tissue to use for Hi-C.
The amount of fixed tissue to use for Hi-C is a pellet/pellets that contains  -  of DNA.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the permeabilization buffer 1 (PB1).  ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 8.0)400 μl(10 mM)35 M NaCl80 μl(10 mM)410% (w/v) NP-40800 μl(0.2%)5H2O38.72 ml 6Total40 ml
This section includes the following steps.Cell/tissue permeabilization - Steps 58 - 83Restriction enzyme digestion - Steps 84 - 86
The number of cells to use for Hi-C is determined based on the amount of DNA.Use 1×106  cells for an animal with the genome size of 3-3.5 Gb (e.g., human and mouse), or 2×106 cells for an animal with the genome size of 1-1.5 Gb (e.g., chicken and western clawed frog).For tissue samples, use a pellet that contains 2-10 μg of DNA.
Follow cells/tissue resuspension steps as written, i.e., vortex mix or pipet mix.
ABC1ReagentAmountFinal Concentration21 M Tris-HCl (pH 8.0)400 μl(10 mM)35 M NaCl80 μl(10 mM)410% (w/v) NP-40800 μl(0.2%)5H2O38.72 ml 6Total40 ml
Filtrate and store at  .
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take ( ×n) + 5% extra volume of PB1 in a new tube, add 1/100 vol of a proteinase inhibitor cocktail (PI), and put on ice.
400 µl
Add PI to PB1 just before use.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take the frozen tissue out from the freezer (from Step 29) and immediately add .
[PB1 (with PI)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Pipet mix and transfer the tissue entirely into a dounce homogenizer pre-cooled on ice.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Homogenize the tissue by ~20 strokes of dounce homogenization on ice with the tight pestle B, then transfer the homogenate into a new microtube (1.5 ml).
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-1000 pipet.
300 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 or a P-200 pipet, and put the sample tube on ice.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the permeabilization buffer 2 (PB2) at .  ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 2.125 μl(1X)310% (w/v) SDS7.5 μl(0.3%)4PI (100X)2.5 μl(1X)5H2O215 μl 6Total250 μl
0 Room temperature
ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 2.125 μl(1X)310% (w/v) SDS7.5 μl(0.3%)4PI (100X)2.5 μl(1X)5H2O215 μl 6Total250 μl
Make fresh every time.Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  in a tube of appropriate size.
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer 2.1]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10% (w/v) SDS]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[PI (100X)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and pipet mix.
[PB2]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate/shake the sample tube in a thermal mixer for  at , .
37 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube.
[20% TritonX-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate/shake the sample tube in a thermal mixer for  at , .
37 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the DNA-preparation buffer.   ABC1ReagentAmountFinal Concentration25 M NaCl250 μl(250 mM)30.5 M EDTA50 μl(5 mM)410% (w/v) SDS500 μl(1%)5H2O4.2 ml 6Total5 ml
ABC1ReagentAmountFinal Concentration25 M NaCl250 μl(250 mM)30.5 M EDTA50 μl(5 mM)410% (w/v) SDS500 μl(1%)5H2O4.2 ml 6Total5 ml
Store DNA-preparation buffer at  .Warm the DNA-preparation buffer at   to dissolve precipitates formed during storage.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  (3%) aliquot (from Step 71) in a new 1.5 ml microtube as a pre-digest DNA control (ctr-1), add  and store at  until the end of DAY2.
8 µl
[DNA-preparation buffer]
-20 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-200 pipet.
200 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 or a P-200 pipet.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare washing buffer (WB) with NEBuffer DpnII (for the DpnII digested sample).  ABC1ReagentAmount (x1 wash cycle)Final Concentration210X NEBuffer DpnII50 μl(1X)320 mg/ml BSA2.5 μl(0.1 mg/ml)420% (w/v) TritonX-1001.25 μl(0.05%)5H2O446.25 μl 6Total500 μl
ABC1ReagentAmount (x1 wash cycle)Final Concentration210X NEBuffer DpnII50 μl(1X)320 mg/ml BSA2.5 μl(0.1 mg/ml)420% (w/v) TritonX-1001.25 μl(0.05%)5H2O446.25 μl 6Total500 μl
Do not use NEBuffer 3.1 for the samples for DpnII digestion.Make WB for HindIII digestion with NEBuffer 2.1 (that contains BSA).Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  in a tube of appropriate size.
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer DpnII]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20 mg/ml BSA]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20% (w/v) TritonX-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet, and put the sample tube on ice.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Prepare the restriction-enzyme mix (for DpnII digestion) on ice.  ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer DpnII20 μl(1X)320 mg/ml BSA1 μl(0.1 mg/ml)4DpnII (50 U/μl)8 μl(400 U)520% Triton X-1000.5 μl(0.05%)6H2O170.5 μl 7Total200 μl
ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer DpnII20 μl(1X)320 mg/ml BSA1 μl(0.1 mg/ml)4DpnII (50 U/μl)8 μl(400 U)520% Triton X-1000.5 μl(0.05%)6H2O170.5 μl 7Total200 μl
Do not use NEBuffer 3.1 for DpnII because non-specific cleavage (star activity) may be induced.Use NEBuffer 2.1 (that contains BSA) for HindIII digestion.For restriction enzymes at different concentration, e.g., HindIII at 100U/μl, adjust the volume with H2O.Make 5% extra volume.
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  in a tube of appropriate size.
[H2O]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[10X NEBuffer DpnII]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20 mg/ml BSA]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[DpnII (50 U/ul)]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add .
[20% Triton X-100]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Add  to the sample tube and pipet mix.
[restriction-enzyme mix]
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Incubate the sample tube in a thermal mixer for ~ at , .
37 °C
?. [Section 4. (DAY 1) Restriction enzyme digestion]
Take  (3%) aliquot in a new 1.5 ml microtube as a digested-DNA control (ctr-2), add  and store at  until the end of DAY 2.
6 µl
[DNA-preparation buffer]
-20 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
This section includes the following steps.DNA fill-in reaction - Steps 88 - 110Ligation reaction - Steps 111 - 113
Follow cells/tissue resuspension steps as written, i.e., vortex mix or pipet mix.
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-200 pipet.
100 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-200 pipet or a P-100 pipet, and put the sample tube on ice.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare the washing buffer (WB) with NEBuffer 2.1.  ABC1ReagentAmount (x1 wash cycle)Concentration210X NEBuffer 2.150 μl(1X)320% (w/v) TritonX-1001.25 μl(0.05%)4H2O448.75 μl 5Total500 μl
ABC1ReagentAmount (x1 wash cycle)Concentration210X NEBuffer 2.150 μl(1X)320% (w/v) TritonX-1001.25 μl(0.05%)4H2O448.75 μl 5Total500 μl
Prepare buffer to perform total of four or two wash cycles (at Steps 93, 98, 105) for the DpnII-digested sample or the HindIII-digested sample respectively.Make 5% extra volume.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[10X NEBuffer 2.1]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% (w/v) TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Repeat the washing cycle (Steps 93 - 97) twice more for the DpnII-digested sample (total of three wash cycles).
Additional washing cycle is not required for the HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare the DNA fill-in mix on ice.  ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 210 μl(1X)30.4 mM biotin-14-dATP3.75 μl(15 μM)41 mM dCTP1.5 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)6 μl(30 U)820% TritonX-1000.25 μl(0.05%)9H2O75.5 μl 10Total100 μl   ABC1ReagentAmountFinal Concentration210X NEBuffer 210 μl(1X)31 mM dATP1.5 μl(15 μM)40.4 mM biotin-14-dCTP3.75 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)3 μl(15 U)820% TritonX-1000.25 μl(0.05%)9H2O78.5 μl 10Total100 μl
ABC1ReagentAmount (x1 reaction)Final Concentration210X NEBuffer 210 μl(1X)30.4 mM biotin-14-dATP3.75 μl(15 μM)41 mM dCTP1.5 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)6 μl(30 U)820% TritonX-1000.25 μl(0.05%)9H2O75.5 μl 10Total100 μl
ABC1ReagentAmountFinal Concentration210X NEBuffer 210 μl(1X)31 mM dATP1.5 μl(15 μM)40.4 mM biotin-14-dCTP3.75 μl(15 μM)51 mM dGTP1.5 μl(15 μM)61 mM dTTP1.5 μl(15 μM)7Klenow DNA polymerase (5 U/μl)3 μl(15 U)820% TritonX-1000.25 μl(0.05%)9H2O78.5 μl 10Total100 μl
Make a DNA fill-in mix that contains either biotin-14-dATP or biotin-14-dCTP, for the DpnII-digested sample or the HindIII-digested sample respectively.Make 5% extra volume.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Take  in a tube of appropriate size.
[H2O]
Take   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[10X NEBuffer 2]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[0.4 mM biotin-14-dATP]
Add   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dCTP]
Add   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dGTP]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[1 mM dTTP]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[Klenow DNA polymerase (5 U/μl)]
Add   for the DNA fill-in mix for HindIII-digested sample.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and pipet mix.
[DNA fill-in mix]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Incubate the sample tube in a thermal mixer for  at , .
25 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and vortex mix.
[WB]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove  of the supernatant using a P-1000 pipet.
400 µl
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time:  - Centrifugation temperature:
4 °C
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Remove the remaining supernatant using a P-100 pipet or a P-200 pipet.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Prepare ligation mix on ice.  ABC1ReagentAmountFinal Concentration2T4 DNA ligase buffer (10X)10 μl(1X)3T4 DNA ligase (2,000 U/ul)2 μl(4,000 CEU)420% TritonX-1000.25 μl(0.05%)5H2O87.75 μl 6Total100 μl
ABC1ReagentAmountFinal Concentration2T4 DNA ligase buffer (10X)10 μl(1X)3T4 DNA ligase (2,000 U/ul)2 μl(4,000 CEU)420% TritonX-1000.25 μl(0.05%)5H2O87.75 μl 6Total100 μl
Prepare T4 DNA ligase buffer in small aliquots and store them at   when the buffer is thawed for the first time.Always use an aliquot that is not thawed and frozen repeatedly.Use 2,000 CEU of T4 DNA ligase for the HindIII-digested sample.Make 5% extra volume.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Take  in a tube of appropriate size.
[H2O]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[T4 DNA ligase buffer (10X)]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[20% TritonX-100]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add .
[T4 DNA ligase (2,000 U/ul)]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Add  to the sample tube and pipet mix.
[ligation mix]
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Incubate the sample tube in a thermal mixer for - at , .
16 °C
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Prepare DNA-extraction mix.  ABC1ReagentAmount (x1 reaction)Final Concentration21M Tris-HCl (pH 8.0)2 μl(10 mM)30.5M EDTA2 μl(5 mM)410% SDS30 μl(1.5%)55M NaCl15 μl(375 mM)620 mg/ml Proteinase K10 μl(1 mg/ml)7H2O141 μl 8Total200 μl
ABC1ReagentAmount (x1 reaction)Final Concentration21M Tris-HCl (pH 8.0)2 μl(10 mM)30.5M EDTA2 μl(5 mM)410% SDS30 μl(1.5%)55M NaCl15 μl(375 mM)620 mg/ml Proteinase K10 μl(1 mg/ml)7H2O141 μl 8Total200 μl
Prepare DNA-extraction mix at  .Make 5% extra volume.
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Take  in a tube of appropriate size.
[H2O]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[1M Tris-HCl (pH 8.0)]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[0.5M EDTA]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[10% SDS]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[5M NaCl]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add .
[20 mg/ml Proteinase K]
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add  to the sample tube (from Step 111) and mix gently.
[DNA-extraction mix]
The total amount of the mixture will be  .
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Add  and  to the control tubes (ctr-1 from Step 73 and ctr-2 from Step 87) and mix gently.
[DNA-extraction mix]
[H2O]
The total amount of the mixture will be  .
?. [Section 6.1. (DAY 2) Hi-C DNA purification]
Incubate the sample tubes in a thermal mixer for ~ at , .
65 °C
Use a thermal mixer equipped with a heated lid to avoid condensation of water on the lid.Alternatively, the incubation can be performed in a heated oven.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Take the sample tube out from the thermal mixer (from Step 117) and let it cool down to .
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[RNase A (10 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Incubate sample tube in a thermal mixer for  at , .
37 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[Proteinase K (20 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Incubate the sample tube in a thermal mixer for  at , .
55 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Take the sample tube out from the thermal mixer, let it cool down to , and proceed with DNA extraction.
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube and mix gently.
[Phenol/Chloroform/Isoamyl alcohol solution]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Transfer ~ of the aqueous phase into a new 1.5 ml microtube.
250 µl
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Prepare TE/NaCl solution.  ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl250 μl(250 mM)40.5 M EDTA (pH 8.0)10 μl(1 mM)5H2O4.69 ml6Total5 ml
ABC1ReagentAmountFinal concentration21 M Tris-HCl (pH 8.0)50 μl(10 mM)35 M NaCl250 μl(250 mM)40.5 M EDTA (pH 8.0)10 μl(1 mM)5H2O4.69 ml6Total5 ml
Store TE/NaCl solution at  .
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the sample tube containing Phenol and mix gently.
[TE/NaCl solution]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
0 Room temperature
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Transfer ~ of the aqueous phase into the microtube containing the first aqueous phase.
300 µl
The total volume of the collected aqueous phase will be ~ .
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the collected aqueous phase and mix gently.
[glycogen solution (20 mg/ml)]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add  to the collected aqueous phase and mix gently until the solution becomes homogeneous.
[2-propanol]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Decant the supernatant, add , and mix gently to rinse the DNA pellet.
[70% EtOH]
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Decant the supernatant.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Centrifuge the sample tube again.Centrifugation speed: Centrifugation time: Centrifugation temperature:
4 °C
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Remove the supernatant completely with a P-100 or a P-200 pipet, and keep the lid open for ~ to allow the ethanol to evaporate.
Do not over dry the pellet.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Quantitate DNA using  of the DNA samples with the Qubit dsDNA High Sensitivity Kit.
1 µl
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
The following procedure should be performed as described in the step-case for the dissociated cells.Start from Section 7. (DAY3) Hi-C DNA QC (QC1), Step 109.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Put the sample tube on ice.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Put the sample tube on ice.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Put the sample tube on ice.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Put the sample tube on ice.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Put the sample tube on ice.
?. [Section 5. (DAY 2) DNA fill-in and ligation]
Put the sample tube on ice.
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add - and  to the Hi-C DNA and the control DNAs (ctr-1 and ctr-2) respectively.
30 µl
[EB]
[EB]
Mix gently to avoid shearing of the DNA.Store DNA samples at   or  .
?. [Section 6.2. (DAY 3) Hi-C DNA purification]
Add - and  to the Hi-C DNA and the control DNAs (ctr-1 and ctr-2) respectively.
30 µl
[EB]
[EB]
Mix gently to avoid shearing of the DNA.Store DNA samples at   or  .