TITLE: Enzymatic fragmentation of plant chromatin for Hi-C libraries
AUTHORS: ignacio.carvajal, Elena Hilario

[DESCRIPTION]
The aim of this protocol is to learn how to prepare, evaluate and optimize a plant chromatin sample to produce a proximity ligated sample ready for NGS library preparation. We introduce several quality checks along the process to avoid wasting precious material or time working with a suboptimal sample (quality and quantity-wise). 


[BEFORE_START]
Characterize your sample

The protocol is developed for plant leaf tissues that will yield at least 5 μg nuclear gDNA per gram of leaf sample. It is crucial that you characterize your biological sample before starting. This will give you a good understanding of the expected yields along the process and make decisions if you fall below the minimum amount of DNA to safely continue to the next stage.
Adjust the amount of starting leaf tissue to obtain the desired amount of ngDNA and make sure you have at least 6 tubes of nuclei before starting because it will take at least two tries before you get the optimal digestion conditions. Each tube should contain at least 2 μg of ngDNA.

How to anneal the bridge adaptor

Dissolve the oligos in TE pH 7.5, at 1 mM. Store at -20°C after use
Heat up a water bath to 65°C, containing at least 3 L of water
Prepare the following mix in a 1.5 mL screw capped tube: 
 
Vortex briefly and do a quick spin
Incubate the adaptor mix at 65°C for 5 min
Transfer  ~1.5 L from the water bath to a shallow plastic container (2 L capacity) on the lab bench
Transfer the adaptor mix tube to the container, and place a thermometer
Let it cool down to ~ 25°C, it should take about 2.5 h, but you can leave it overnight
When ready, do a quick spin and store at -20°C
This solution is enough for five bridge ligation reactions

Crosslinking Solution

0.5 mL 1X PBS + 27 µL  , freshly prepared


Streptavidin magnetic bead binding buffers

The following buffers can be prepared ahead of time and stored at 4 °C  for at least 6 months
 



[GUIDELINES]
Every sample brings a different challenge and no chromatin protocol is universal. It is recommended to practice with a less precious sample before attempting the preparation of the final Hi-C library. There are a few commercial kits available to prepare Hi-C libraries and perform well under most circumstances, especially since often they have good technical support to help you along the way. This protocol describes an in vitro enzymatic fragmentation of plant chromatin and aims to help understand the importance of QC steps along the process.

Ideally, the chromatin fragmentation should be random, using DNase I, but in practice, this approach can take some time to optimize and if your sample size is limited you may consider using a 4-base restriction enzyme, selected based on the genome's GC content. Or you could also use a mix of two or more 4-base restriction enzymes to target as many sites as possible, either by having a different restriction site or a different methylation sensitivity. Although it is easier to control a restriction enzyme digestion, beware that the genome coverage is reduced, compared to a randomly fragmented chromatin sample. We strongly recommend to perform digestion tests on nuclear gDNA preps with several restriction enzymes until you find a combination that will produce an even digestion profile.

Using a biotinylated bridge to mark the contact ends adds extra steps and some assurance that the contacts detected are real, but you could also omit this step and fill in the 5'-end overhangs with biotinylated dNTPs. Beware the length of the carbon chains that links the biotin to the nucleotide (the standard is 6 carbon atoms, used in this protocol) has an impact on how well it will bind to the streptavidin molecule. The longer the carbon chain, the more efficient the binding.

If you have trouble isolating a clean nuclei pellet, you could purify it with a Percoll gradient, as described here. The wash volumes of the chromatin bound to magnetic beads can be increased if the sample is hard to clean, or extra washes can be implemented too. The lysis buffer volume can also be increased to reduce the amount of contaminants even before starting the chromatin washes.

[STEPS]
SECTION: Nuclei isolation and integrity check
1. 

 
Add 20 mL  to the ground tissue by gently dislodging it with an inoculation loop. Stir gently and drag the lump up against the tube wall until it is resuspended
SECTION: Nuclei isolation and integrity check
2. Centrifuge 3500 rpm, 5 min, 10 °C. Pour off the supernatant into designated β-ME-WASTE container. The supernatant will have the consistency of light syrup and it might be light green with fine sediment
SECTION: Nuclei isolation and integrity check
3.     at least another 2 times. It can take up to 4 rounds of washing to obtain a supernatant that pours off like water and is clear. Place the tube on ice
SECTION: Nuclei isolation and integrity check
4. Add 20 mL and gently resuspend the ground tissue by tapping the bottom of the tube and mixing by inversion. Add another 20 mL and mix by inversion. Place the tube horizontally over an ice bucket, and on the orbital   
SECTION: Nuclei isolation and integrity check
5. Assemble the funnel over a 50 mL Falcon tube and place the Miracloth. Pre-wet the miracloth with some NEB complete buffer
SECTION: Nuclei isolation and integrity check
6. Filtrate the extract through one sheet of Miracloth
SECTION: Nuclei isolation and integrity check
7. Filtrate the extract through a 100 µm cell strainer assembled over a 50 mL Falcon tube
SECTION: Nuclei isolation and integrity check
8. Filtrate the extract through a 40 µm cell strainer assembled over a 50 mL Falcon tube. Lift the cell strainer slightly and press the mesh against the tube inner wall to speed up the filtration
 

SECTION: Nuclei isolation and integrity check
9. Collect the nuclei by centrifugation at 1800 x g, 15 min, 10 °C 
SECTION: Nuclei isolation and integrity check
9.1. Discard the supernatant in the designated β-ME-WASTE container
SECTION: Nuclei isolation and integrity check
10. Add 25 mL and resuspend the pellet gently by tapping the bottom of the tube and mixing by inversion

SECTION: Nuclei isolation and integrity check
11.     
SECTION: Nuclei isolation and integrity check
12. Add 1 mL and detach the pellet gently by tapping the bottom of the tube. Resuspend the nuclei using a 1 mL pipette set at 500 μL and wide bore tips by drawing the liquid in and out very slowly. Avoid introducing air bubbles. It is very important the nuclei suspension is homogeneous
 

SECTION: Nuclei isolation and integrity check
13. Split the nuclei suspension in eight 1.5 mL screw capped tubes, spin down 1 min at maximum speed, discard the buffer and the nuclei pellet tubes at -80 °C
 

SECTION: Chromatin Crosslinking
14. Add 0.5 mL to one of the defrosted nuclei tubes  . Tap gently the bottom of the tube, or use an inoculation loop to dislodge the nuclei from the tube walls. The nuclei should be fully resuspended
SECTION: Chromatin Crosslinking
15. Add 0.5 mL to the nuclei pellet, and mix by inversion
SECTION: Chromatin Crosslinking
16. Incubate at 10 min with gentle mixing using the orbital shaker by placing the tube horizontally
 

SECTION: Chromatin Crosslinking
17. Centrifuge at 3500 rpm, 5 min and discard the supernatant in designated FA-WASTE container
 

SECTION: Chromatin Crosslinking
18. Quench the crosslinking reaction by adding 1 mL and resuspend the pellet gently by tapping the bottom of the tube, or by using an inoculation loop, and mix by inversion
SECTION: Chromatin Crosslinking
19. Centrifuge at 3500 rpm, 5 min and discard the supernatant in designated FA-WASTE container
 


SECTION: Lysate characterization
20. Resuspend nuclei in 500 µL and incubate on the thermal mixer set at   

 

SECTION: Lysate characterization
21. Take  50 µL  and   to extract the lysate DNA and run 100 or 200 ng in a 1% agarose gel.  The DNA should be > 20 kbp. Do not proceed if the DNA is degraded.

Keep the rest of the extracted lysate DNA at 4ºC for quality check in the Fragment Analyzer at a later stage, together with the DNase I titration experiment (see below).

 

SECTION: Chromatin Capture
22. Calculate the volume of lysate to have 2 μg of DNA    and add 2 volumes of  AMPure XP beads, mix and let it stand 5 min
 

SECTION: Chromatin Capture
23. Place the tube on the magnet for at least 2 min or until the solution is clear.
Discard the supernatant, and wash the beads with 1 mL.
Place the tube on the magnet again for at least 2 min or until the solution becomes clear and discard it
SECTION: Chromatin Capture
24.   at least one more time, but it might take 4 or more rounds of washing until the solution becomes clear when the beads are on the magnet. The total number of washes depend on the plant species.
 

SECTION: DNase I Digestion Dilution Series
25. 
This section explains how to determine the optimal condition to digested chromatin. You will use one of the the nuclei tubes   .
Once you have found the optimal condition, you will apply to them to a new nuclei tube (section DNase I Digestion and onwards)

Prepare the following solutions:

 

SECTION: DNase I Digestion Dilution Series
26. Mix the DNase I 1 U/µL stock by inversion 20 times, quick spin
Mix by inversion again 20 times, and quick spin
SECTION: DNase I Digestion Dilution Series
27. After removing the last 1X Wash buffer solution from   add 500 µL   and resuspend by vortexing briefly
SECTION: DNase I Digestion Dilution Series
29. Transfer 50 µL from   to the tube labelled T = 0 h
Transfer 100 µL to each tube A-1 h to D-1 h
Place the 5 tubes on ice
SECTION: DNase I Digestion Dilution Series
30. Start the digestion by adding 10 μL of its corresponding dilution to tubes A-1 h to D-1 h and flick the bottom of the tube to mix the beads
Transfer the 5 tubes to the thermomixer 
60 min 
SECTION: DNase I Digestion Dilution Series
31. Stop the reaction by adding  25 µL to tubes A-1 h to D-1 h and vortex 3 s
Add 12.5 µL to tube T = 0 h and vortex 3 s
SECTION: DNase I Digestion Dilution Series
32. Add 200 µL to each tube, vortex briefly and place them on the magnet until the solution is clear
SECTION: DNase I Digestion Dilution Series
32.1. Discard the supernatant in the MnCl2 -WASTE container and   to repeat the wash step
SECTION: DNase I Digestion Dilution Series
33. After removing the supernatant, add 200 µL and 2 µL to each tube, vortex briefly and store at 4 °C


SECTION: Chromatin Quality Check
34. 
Incubate 1 mL  for 55 °C before use
Vortex and quick spin before opening the tube
SECTION: Chromatin Quality Check
36. Add 100 µL to each tube
SECTION: Chromatin Quality Check
37. Place the tubes in the thermomixer   and start the program
SECTION: Chromatin Quality Check
38. When the Proteinase K digestion is finished, quickly spin down the tubes and place them on the magnet for at least 2 min
SECTION: Chromatin Quality Check
39. Transfer the supernatant containing the DIGESTED CHROMATIN to a new tube and discard the beads
SECTION: Chromatin Quality Check
40. Add 180 µL, mix by flicking the bottom of the tube until the solution is homogeneous
Let it stand 5 min
SECTION: Chromatin Quality Check
40.1. Capture the beads with the magnet for at least 2 min and discard the supernatant in the MnCl2-WASTE container

SECTION: Chromatin Quality Check
40.2. Without removing the tube from the magnet add 200 µL over the beads and wait for exactly  30 s before removing the ethanol
Repeat the ethanol wash one more time
SECTION: Chromatin Quality Check
40.3. Spin down the tubes 2 s and place them on the magnet again and remove all the liquid
SECTION: Chromatin Quality Check
41. Add 50 µL directly onto the beads, close the tube and remove from the magnet. Mix the beads by gently flicking the bottom of the tube
Do a quick spin (~ 1 second) to collect the beads at the bottom
Let the DNA elute at least 10 min 
SECTION: Chromatin Quality Check
41.1. Place the tube on the magnet for 2 min until all the beads are against the magnet and transfer the eluted DNA to a new tube. Keep at 55 Room temperature  
 

SECTION: Chromatin Quality Check
34.1. Set the thermomixer program as follows:
55°C 15 min 1250 rpm → 68°C 45 min 1250 rpm
SECTION: DNase I Digestion Dilution Series
25.1. Set the thermomixer at    ∞ min
SECTION: Chromatin Quality Check
42. Quantify 1 µL with the High Sensitivity dsDNA Qubit kit and estimate the total amount of DNA for each time point (see table below)
SECTION: Chromatin Quality Check
43. Prepare the samples for the Fragment Analyzer run. Use the HS NGS kit 1-6000 bp:

If the concentration is > 5 ng/µL, dilute it to 2.5 ng/µL with TE buffer and use 2 µL 
If the concentration is < 2.5 ng/μL use 2 µL.
Open the Fragment Analyzer outfile with ProSize 3.0 and perform the Smear Analysis by selecting the following fragment ranges, and fill out the table below:
SECTION: Chromatin Quality Check
44.  
           % Smear (bp)  Qubit              100-2500  300-2000  100-300  2000-2500  ng/uL  Vol µL  Yield ng       Optimal  Middle  Low end  High end             Nuclei sample            50       Lysate            10       Zero            20        5 min 0.02 U            20        10 min 0.02 U            20        15 min 0.02 U            20        20 min 0.02 U            20        25 min 0.02 U                 20     

 

SECTION: Chromatin Quality Check
45. The amount of DNA in the nuclei sample allows you to estimate the DNA yield per tube and per gram of tissue
The lysate should produce intact DNA, with same profile as your nuclei QC sample described in  . The lysate QC step allows you calculate how much chromatin to expect per lysis event. The lysate and T = 0 h are internal controls to monitor if DNA degradation occurred during sample manipulation
The goal of the DNase I dilution series is to determine the optimal amount of DNase I where most of the DNA is within the 100-2500 bp range

≥ 50% of the input DNA is within 100-2500 bp

If you have at 0.9-1 μg of optimally digested chromatin you can now take another nuclei tube and process it in the same way (Section DNase I onwards) to produce either a Nanopore or an illumina library
If you have ~ 150 ng of optimally digested chromatin you can only prepare an illumina library under these condition
If you have 80-100 ng of optimally digested chromatin you are at the lower limit for preparing an illumina library, but it is till possible to produce one

< 50% of the input DNA is within 100-2500 bp

If most of the smear is ≤ 300 bp for all dilutions (or time points - see step case option), the sample was over digested. Repeat the DNase I dilution series but incubate the digestion for 30 min only. If you did a time series   use a 0.005 U/μL dilution of DNase I

If most of the smear is ≥ 2500 bp, the sample was under digested. Before repeating or modifying the chromatin prep, check that the DNase I stock is working correctly. Perform a control digestion of 1 µg of Lambda DNA, 20 min only, in 1X DNase I buffer + 10 mM MnCl2, 0.02 U DNase I, in a total volume of 20 μL, 37ºC. Stop the reaction with 1 μL 0.5 M EDTA pH, add 4 μL 6X gel loading buffer and load it all in a 1% agarose gel and include an aliquot of undigested Lambda DNA as control (1X TAE, 140 V, 30 min, stain the gel with SybrSafe after the run or add it directly on the gel). If the test shows no digestion, buy a new DNase I stock (with MnCl2)

If the under digested result is accompanied by a high DNA yield (>5 µg), QC the lysate again and make sure you only use 2 μg for the chromatin capture step
SECTION: DNase I Digestion
46. This section describes how to process one nuclei tube from crosslinking to the DNase I digestion step once the optimal reaction conditions have been established.

You will collect samples of lysate, T = 0 h and optimal digestion time for QC later. This is a precaution to ensure the same results observed earlier are reproduced
SECTION: End Repair
47. 
From the Optimal QC sample determine how much DNA is digested and captured in the AMPure XP beads
The NEBNext Ultra II End repair can only repair up to 1 μg of DNA. If there is more than 1 µg of digested chromatin needs to be repaired, up-scale the End Repair according to the table shown below
The NEBNext FFPE DNA repair reaction is only needed for Nanopore library preparation and can only repair up to 1 μg of DNA. If you are preparing an Illumina library, omit this reagent
Make sure the buffers are fully defrosted and dissolved. Vortex and quick spin all the reagents
SECTION: End Repair
48. Place the digested captured chromatin from   on the magnet and discard the clear supernatant. Wash the beads twice with 200 µL and discard all the liquid
SECTION: End Repair
49. Add the following reagents of the desired library to be prepared to the washed beads in the order shown below. Mix the contents before and after adding the enzymes:

 

SECTION: Bridge Ligation
50. Defrost the ligase buffers and vortex until no solid precipitate (DTT) and no syrupy pellet (polyethylene glycol) are visible. Keep the buffers on ice
SECTION: Bridge Ligation
51. Add the following reagents to the beads in the order specified:

 

SECTION: Proximity Ligation
52. While performing the Bridge ligation, defrost the ligase buffers and vortex it until no solid precipitate (DTT) and no syrupy pellets are visible. Keep the buffers on ice
SECTION: Proximity Ligation
53. Add the following reagents to the beads   in the order specified:

 

SECTION: Chromatin Reverse Crosslinking and Quality Check
54. 
Program the thermomixer as follows: 55°C 15 min 1250 rpm → 68°C 45 min 1250 rpm
Prewarm the thermomixer at 55°C for ~ 15 min without shaking
SECTION: Chromatin Reverse Crosslinking and Quality Check
55. Incubate 1 mL for 10 min at 55 °C before use
Vortex and quick spin before opening the tube
SECTION: Chromatin Reverse Crosslinking and Quality Check
55.1. Quick spin the  , place it for at least 2 min and discard all the liquid

SECTION: Chromatin Reverse Crosslinking and Quality Check
55.2. Add 100 µL and vortex briefly
SECTION: Chromatin Reverse Crosslinking and Quality Check
55.3. Add 1 µL, vortex briefly and quick spin the tube (see note  )

SECTION: Chromatin Reverse Crosslinking and Quality Check
55.4. Place the tube in the thermomixer and start the program   
SECTION: Chromatin Reverse Crosslinking and Quality Check
56. Quick spin the tube and TRANSFER THE LIQUID TO A NEW TUBE

THIS IS YOUR INTRA-MOLECULAR LIGATED CHROMATIN (IMLC)

 
SECTION: Chromatin Reverse Crosslinking and Quality Check
56.1. Add 50 µL to the beads, vortex gently, place for 2 min and transfer the liquid to the rest of the IMLC from the previous step
The total volume is 150 µL IMLC.  Discard the beads

 

SECTION: Chromatin Reverse Crosslinking and Quality Check
57. Add 105 µL to the 150 μL IMLC and vortex gently until the sample is homogeneous

 

SECTION: Chromatin Reverse Crosslinking and Quality Check
57.1. Incubate  5 min 
SECTION: Chromatin Reverse Crosslinking and Quality Check
57.6. Add 54 µL directly onto the beads, vortex gently and do a quick spin
Let the DNA elute at room temperature for at least 10 min

SECTION: Chromatin Reverse Crosslinking and Quality Check
58. Quantify 1 µL and also 1 μL of T = 0 h and Optimal QC samples   with the HS dsDNA Qubit kit
Run 2 µL on the Fragment Analyzer (HS NGS kit 1-6000 bp)
SECTION: Chromatin Quality Check
35. Equilibrate the tubes   at room temperature ~ 5 minutes
Place the tubes on the magnet for at least  2 min until the solution becomes clear
Discard the supernatant and place the tubes on a rack
SECTION: Nuclei isolation and integrity check
13.1. It is recommended to check the DNA integrity before preparing the library

Add 50 µL to the nuclei    vortex briefly
Add  1 µL and vortex 2 seconds
Place the tube in the thermomixer programmed as follows:
55°C 15 min 1250 rpm → 68°C 45 min 1250 rpm
Spin down the tube briefly and add 100 µL  mix and let stand 5 min 
Place 2 min  or until the solution is clear. Discard the supernatant
While the tube is on the magnet, add 200 µL  over the beads and remove it 30 s. Repeat this step one more time
Quick spin the tube, place it on the magnet and remove all ethanol
Remove tube from magnet and add 50 µL. Tap the bottom of the tube to resuspend the beads and let the DNA elute at least 10 min
Place the tube on the magnet and transfer the eluted DNA to a new tube
Quantify 1 µL with the HS dsDNA Qubit kit
Calculate the amount of nuclear genomic DNA in 50 μL and then extrapolate to   
Calculate how much ngDNA is per tube (~118 μL if split in 8 tubes, or ~158 μL if split in 6 tubes). Ideally you will have ≥3 μg of ngDNA per tube
Analyze 200 ng in a 1% agarose gel against the 1 kb+ ladder and 200 ng. The extracted DNA should be >20 kbp. If the DNA is not intact, do not proceed with crosslinking and DNase I test. Prepare a new nuclei prep and QC the sample again
SECTION: Nuclei isolation and integrity check
12.1. Transfer 50 µL to a 1.5 mL tube to check the DNA integrity (see below)
SECTION: DNase I Digestion Dilution Series
26.1. Transfer 20 μL of DNase I solution to a new tube and add 5 μL DNase I 1 U/μL stock
Mix by inversion 20 times and quick spin
The DNase I concentration of this dilution is 0.2 U/μL
Place tube on ice
SECTION: Chromatin Quality Check
36.1. Add 1 µL to each tube, vortex briefly and quick spin
 

SECTION: Nanopore library preparation
59. To prepare a Nanopore library you will need a least 1 µg. Check the Nanopore Community page to select the kit and best conditions for sequencing
SECTION: Chromatin Reverse Crosslinking and Quality Check
58.1. Store the IMLC at -20 °C until ready to prepare the sequencing library
SECTION: Illumina library preparation
60. IMLC End Prep and Illumina adaptor ligation

The universal illumina adaptor and USER enzyme can be found in the NEBNext Multiplex Oligos for Illumina (Index Primers Set 3) kit

Set up the following reaction in a 200 μL PCR tube:

 
Place the tube on a thermocycler programmed as follows: 20°C 30 min → 65°C 30 min → 12°C ∝
SECTION: Illumina library preparation
61. Add 2.5 µL, vortex and quick spin
SECTION: Expected results
68. We have tested this protocol on several species and although not all of them showed ideal digestions and IMLC profiles (see step case below) the MiSeq QC run produced usable libraries.

 
The sea urchin and bat samples included in this section were grounded in liquid nitrogen, washed and resuspended in 1X PBS before crosslinking. After quenching the formaldehyde with 1X Wash buffer, the cells were filtered through a 200 μm cell strainer and collected by centrifugation. The myna bird blood cells were defrosted in 1X TE pH 7.5, 100 mM EDTA as soon as they were taken out of the -80ºC freezer, spun down, washed with 1X PBS twice before crosslinking and processed as the other animal samples.

The table below shows the expected yields along the protocol across 5 different plants.

 
It is crucial to use no more than 100 ng of IMLC-illumina sample for the streptavidin magnetic bead capture because not all of it will be captured. Under the conditions described in this method, about 50 ng of the initial 100 ng of IMLC-illumina library is bound to the streptavidin beads. It is possible that by using a longer linker connecting the biotin hapten to the Bridge (+) strand, the steric hindrance might be avoided.

The Hi-C library amplification double size selection might look a bit lumpy:

 
And the next table shows the metrics obtained with the MiSeq QC run:   
SECTION: Illumina library preparation
62. Add the following reagents to the End prep IMLC + illumina adaptor tube in the order specified:

 

SECTION: Illumina library preparation
62.1. Incubate at 20 °C. Or leave it in the refrigerator overnight

SECTION: Illumina library preparation
62.2. Add 3 µL, vortex, quick spin and incubate at 37 °C 

SECTION: Illumina library preparation
62.3. Spin down the tube and transfer all the sample to a 1.5 mL tube
SECTION: Illumina library preparation
62.4. Add 50 µL to the PCR tube, vortex, quick spin and transfer all to the 1.5 mL tube
Total volume: 153 μL
SECTION: Illumina library preparation
63. Add 120 µL and vortex gently until completely homogeneous
SECTION: Illumina library preparation
63.1. Incubate Room temperature 
Place the tube on magnet 2 min or until the solution becomes clear and discard the solution
SECTION: Illumina library preparation
63.3. Add 100 µL to the tube, remove from the magnet, and resuspend the beads by gently vortexing the tube
Incubate Room temperature for at least 10 min 
Return the tube to the magnet and when the solution becomes clear transfer it to a new 1.5 mL tube
This is now your IMLC-illumina library
SECTION: Illumina library preparation
63.2. Add 180 µL to the tube while on the magnet
Discard all liquid after 30 s 
Repeat this wash one more time
Quick spin the tube, place it on the magnet and remove all liquid
SECTION: Illumina library preparation
64. Quantify 1 µL with the Qubit HS dsDNA kit
 

SECTION: Illumina library preparation
65. The following steps describe how to prepare the streptavidin magnetic beads for binding an IMLC-illumina library at a concentration of 2.5 ng/μL or higher.

If your IMLC-illumina library is less concentrated, resuspend the SAMB in a different volume. For example: The IMLC-illumina library is at 1.5 ng/μL. To have 100 ng for SAMB capture you need 66 µL. Resuspend the prepared SAMB beads in 66 µL instead of 40 μL as described here:

Streptavidin magnetic bead (SAMB) preparation

Coat one 1.5 mL screw-capped tube by adding 1 mL, mix and remove all liquid
Add 1 mL to the tube
Vortex the SAMB until fully resuspended and transfer 20 µL to the tube, mix by pipetting. This volume contains 200 μg of beads
Place the tube on the magnet for at least 2 min and discard half of the liquid
Quick spin the tube and place it on the magnet
When the solution is clear, discard it all
Remove the tube from the magnet and add 20 µL and mix by pipetting
Place the tube on the magnet for 2 min and discard the liquid
Repeat this wash step two more times
Add 40 µL and mix by pipetting
SECTION: Illumina library preparation
67. Library amplification and size selection

Set up the following PCR master mix:

 
Vortex until the beads are mixed evenly
Aliquot 50 µL per PCR tube and place them in the thermocycler

PCR profile:

98ºC 30 sec → (98ºC 10 sec - 65ºC 75 sec) x 12 → 65ºC 5 min → 12ºC ∞
SECTION: Illumina library preparation
66. SAMB capture

Calculate the volume required of IMLC-illumina library   to have 100 ng and adjust it to a final volume of 40 µL. Mix and quick spin the tube
Transfer the IMLC-illumina solution to the prepared SAMB  , vortex briefly and place in the thermomixer 1250 rpm, 30 min, 25 °C 
Quick spin the tube and place it on the magnet for at least 2 min and discard the solution*
Remove the tube from the magnet and add 120 µL and mix by pipetting
Place the tube on the  magnet for at least 2 min and discard the solution*
 
6. Repeat steps 4 and 5 one more time
7. Remove the tube from the magnet and add 120 µL and mix by pipetting
8. Place the tube on the  magnet for at least 2 min and discard the solution*
9. Repeat steps 8 and 9 one more time
10. Resuspend the beads in 40 µL

The bead concentration is ~ 5 μg beads/μL. Store at 4 °C
The sample is stable at this temperature for at least 2 weeks

SECTION: Illumina library preparation
67.1. Spin down the tubes briefly and transfer all the solution to one 1.5 mL tube
This tube contains the amplified Hi-C library plus IMLC-illumina-SAMB
SECTION: Illumina library preparation
67.2. Wash the PCR tubes:
Add 100 µL to one of the PCR tubes and set the pipette volume to 150 μL
Pipette the solution several times and transfer to the next tube. Repeat this step on all the PCR tubes
Transfer the solution to the 1.5 mL tube  
SECTION: Illumina library preparation
67.4. Add 0.8 - to the PCR reaction (step 67.3.2). For example: if 400 μL were recovered, add 320 μL of AMPure XP beads
Vortex briefly until the solution is homogeneous
 

SECTION: Illumina library preparation
67.5. Incubate at Room temperature 
Place the tube on the magnet for at least 2 min or until the solution is clear
Discard the solution
SECTION: Illumina library preparation
67.6. While still on the magnet, add 500 µL to the beads and remove it exactly after  30 s 
Repeat this step one more time
SECTION: Illumina library preparation
67.7. Quick spin the tube and place it on the magnet again
Remove all traces of ethanol
Remove the tube from the magnet and place it on a rack
Add 100 µL  and gently vortex the tube until the beads are resuspended
Incubate Room temperature 
Place the tube on the magnet and when the solution is clear, transfer it to a new 1.5 mL Eppendorf tube

This is the amplified Hi-C library cleaned with 0.8X AMPure XP beads
SECTION: Illumina library preparation
67.8. Qubit 1 µL with Qubit HS dsDNA kit
Prepare 10 μL of a 2.5 ng/μL dilution of the amplified library and run it in the Fragment Analyzer (HS NGS 1-6000 bp method)

SECTION: DNase I Digestion Dilution Series
28. Prepare one tube labelled T = 0 h and four tubes as follows:
 

SECTION: DNase I Digestion
46.1. Select the time point that contains ≥50% of the DNA within 100-2500 bp to prepare your library
Take one tube    from the -80ºC freezer and perform the steps from   up to  
SECTION: Chromatin Reverse Crosslinking and Quality Check
57.2. Place the tube on magnet and let it stand for at least 2 min or until all the beads are against the magnet and discard the liquid
SECTION: Chromatin Reverse Crosslinking and Quality Check
57.3. While the tube is still on the magnet, add 200 µL 
Remove it exactly after 30 s
SECTION: Chromatin Reverse Crosslinking and Quality Check
57.4. Repeat step   one more time
SECTION: Chromatin Reverse Crosslinking and Quality Check
57.5. Place the tube on the centrifuge and do a quick spin (~ 2 s) and place on magnet again Remove all traces of ethanol
SECTION: Chromatin Reverse Crosslinking and Quality Check
57.7. Place the tube on the magnet for ~ 2 min until all the beads are collected
Transfer the eluted DNA to a new tube
SECTION: DNase I Digestion
46.2. Carry out the DNase I digestion with two time points only: T = 0 h and Optimal QC as follows:

Transfer 50 µL resuspended in  500 µL to a tube labelled T = 0 h
Start the reaction by adding the    to the remaining 450 µL and place both tubes in the thermomixer

For example: if the optimal digestion was observed with dilution B

 


When the time is up, transfer 50 µL to a tube labelled Optimal QC and add 12.5 µL, mix and leave it on a rack at room temperature
Add 12.5 µL  to the T = 0 h, mix and leave it on the rack
Add 100 µL  to the remaining 400 μL of digested chromatin, mix and leave it on the rack
SECTION: DNase I Digestion
46.3. Add 800 µL  to each tube, mix and place on the magnet for at least 2 min  until the solution is clear
Discard all the solution in the MnCl2-WASTE container
Remove the tubes from the magnet and add 1 mL to each tube, mix and place on the magnet again
When the solution is clear transfer it to the MnCl2-WASTE container
SECTION: DNase I Digestion
46.4. Extract the DNA from  T = 0 h and Optimal QC tubes as described before    

These samples, together with the leftover lysate ( ) and the intra molecular ligated chromatin will be quantified and loaded in the Fragment Analyzer later (see Chromatin Reverse Crosslinking Section and Quality Check section below)
SECTION: End Repair
49.1. Place the tube in the thermal mixer set at    
SECTION: End Repair
49.2. Quick spin the tube and place it on the magnet for ∼ 2 min and discard all the liquid
SECTION: End Repair
49.3. Remove the tube from the magnet and add 1 mL and gently resuspend the beads by tapping with a pen
Quick spin the tube and place it for at least 2 min and discard all the liquid
SECTION: End Repair
49.4. Repeat   one more time and proceed to the Bridge Ligation step immediately
SECTION: Bridge Ligation
51.1. Place the tube in the thermomixer set at    
SECTION: Bridge Ligation
51.2. Quick spin the tube and place it for at least 2 min and discard all the liquid
SECTION: Bridge Ligation
51.4. Repeat    one more time making sure all the liquid is discarded
Place the tube on a rack and continue with Proximity Ligation section
SECTION: Bridge Ligation
51.3. Remove the tube from the magnet and add 500 µL and gently resuspend the beads by tapping with a pen
Quick spin the tube and place it for at least 2 min and discard all the liquid
SECTION: Proximity Ligation
53.1. Place the Proximity Ligation reaction tube in the thermomixer set at  

SECTION: DNase I Digestion Dilution Series
26.2. Prepare the dilution series as follows:
 
Mix the dilution by tapping the bottom of the tube 20 times and quick spin
Place on ice
SECTION: Illumina library preparation
67.3. Recover the SAMB-illumina bound library:

Place the 1.5 mL tube   on the magnet for at least 2 min 
Transfer the clear solution to a new 1.5 mL tube and set aside. This is your amplified Hi-C library to be sequenced
Add 50 µL to the SAMB beads, remove it from the magnet, vortex gently and place it in the magnet again for at least  2 min 
Transfer the solution to the 1.5 mL tube set aside earlier (step 67.3.2)
Add 10 µL to the SAMB-illumina bound library and store at 4 °C This sample can be used again to prepare more amplified library if needed, but use it within 1 week
Estimate the total volume of amplified library using a 1000 μL pipette . Expect ~ 400 μL (250 μL PCRs + 100 μL PCR tubes wash + 50 μL beads wash)
SECTION: DNase I Digestion
46.5. Add 200 µL Chromatin Quality Check and 2 µL Chromatin Quality Check to the Digested chromatin tube and store at 4 °C
This is the sample to be used for preparing your library (see section End Repair)
SECTION: Acknowledgements
69. We would like to thank Ashley Jones (Australian National University, Canberra) for joining us in our first Hi-C marathon in 2022, and our colleagues for providing samples to test: Susan Thomson, Wendy Hall, Ed Morgan (Plant and Food Research, New Zealand), Anna Santure (University of Auckland, New Zealand), Annabel Whibley and Darrell Lizamore (Bragato Institute), Jessie Prebble (Landcare Research, New Zealand), Niel Gimmel and Joanne Gillum (University of Otago, New Zealand).