TITLE: s3-GCC
AUTHORS: Andrew    Adey, Ryan     Mulqueen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">s3GCC (symmetrical strand single-cell combinatorial indexing; genome conformation capture) protocol for the capture of Hi-C like chimeric reads, generated through proximity ligation, as well as standard genomic reads.</div><div class = "text-block"><span>See </span><a href="https://mulqueenr.github.io/schic" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://mulqueenr.github.io/schic/</span></a><span> for more information.</span></div></div>

[STEPS]
?. [Prepare oligoes and transposases]
Attached is a s3 molecular layout per step. As well as oligonucleotides used in the protocol. Prior to starting, iTSM plate should be prepared following the protocols listed within this step.Tabs with oligonucleotides to be ordered and prepared:iTSM sci_WGA14_ME_LNAPCR_i5 primersPCR_i7 primers
?. [Prepare oligoes and transposases]
For generation of Tn5 enzyme.
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?. [Prepare oligoes and transposases]
For loading Tn5 enzyme. Load only a single oligonucleotide + mosaic end reverse compliment oligonucleotide per well.Load to a final concentration of equimolar 2.5uM oligonucleotide:mosaic end reverse compliment with 2.5uM Tn5.
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?. [Prepare Buffers]
Construct 50mL Nuclei Isolation Buffer (NIB-Tris):  ABC1Final ConcentrationStock ConcentrationVolume of Stock210 mM Tris HCl, pH 7.41M Tris-HCl, pH7.4500 uL310 mM NaCl5M NaCl100 uL43mM MgCl21M MgCl2150 uL50.1 % Igepal10% Igepal500 uL60.1 % Tween10% Tween500 uL7ddH20to 50mL (add 48.25mL)Store NIB on ice throughout nuclei dissociation and preparation of tagmentation plates.
ABC1Final ConcentrationStock ConcentrationVolume of Stock210 mM Tris HCl, pH 7.41M Tris-HCl, pH7.4500 uL310 mM NaCl5M NaCl100 uL43mM MgCl21M MgCl2150 uL50.1 % Igepal10% Igepal500 uL60.1 % Tween10% Tween500 uL7ddH20to 50mL (add 48.25mL)
OPTIONAL: To prevent protease degradation, we also add 2 tables of Pierce Preotease Inhibitor Tablets, EDTA-Free to NIB following construction. We then vortex to fully dissolve tablets.
NIB is stable at    for at least 1 month without noticable degradation in library quality or nuclei dissociation ability.
?. [Prepare Buffers]
Construct 50mL Nuclei Isolation Buffer (NIB-HEPES):  ABC1Final ConcentrationStock ConcentrationVolume of Stock210 mM HEPES-KOH, pH 7.21M HEPES-KOH, pH 7.2500 uL310 mM NaCl5M NaCl100 uL43mM MgCl21M MgCl2150 uL50.1 % Igepal10% Igepal500 uL60.1 % Tween10% Tween500 uL7ddH20to 50mL (add 48.25mL)Store NIB on ice throughout nuclei dissociation and preparation of tagmentation plates.
ABC1Final ConcentrationStock ConcentrationVolume of Stock210 mM HEPES-KOH, pH 7.21M HEPES-KOH, pH 7.2500 uL310 mM NaCl5M NaCl100 uL43mM MgCl21M MgCl2150 uL50.1 % Igepal10% Igepal500 uL60.1 % Tween10% Tween500 uL7ddH20to 50mL (add 48.25mL)
OPTIONAL: To prevent protease degradation, we also add 2 tables of Pierce Preotease Inhibitor Tablets, EDTA-Free to NIB following construction. We then vortex to fully dissolve tablets.
NIB is stable at    for at least 1 month without noticable degradation in library quality or nuclei dissociation ability.
?. [Prepare Buffers]
Construct 5mL of 10X NEBuffer 2.1  ABC1  Concentration (10X)    Stock Concentration  Volume of Stock2  500mM NaCl    5M NaCl  500 uL3  100mM Tris-HCl  1M Tris HCl (pH 7.9)500 uL4  100mM MgCl2    1M MgCl2  500 uL5  1ng/uL BSA    100ng/uL BSA  50 uL6  pH 7.9 @ 25C    H2O  3450 uL
ABC1  Concentration (10X)    Stock Concentration  Volume of Stock2  500mM NaCl    5M NaCl  500 uL3  100mM Tris-HCl  1M Tris HCl (pH 7.9)500 uL4  100mM MgCl2    1M MgCl2  500 uL5  1ng/uL BSA    100ng/uL BSA  50 uL6  pH 7.9 @ 25C    H2O  3450 uL
?. [Dissociate Adherent Cells]
Different isolation techniques are h ighlighted across the s3 protocol family and can be used interchangeably for downstream processing. For s3WGS we primarliy used adherent cultured cell lines.
NoteIsolation of nuclei is dependent on the sample being used. And optimization should be performed. Below is an example of a nuclei isolation protocol to act as general use for primary brain tissue samples. Other tissues should follow a similar dounce homogenization protocol.
?. [Dissociate Adherent Cells]
Plate 1 million cells in T25 flask the day prior and harvest while cells are subconfluent.
?. [Dissociate Adherent Cells]
Wash adherent cell linesWashed cells in flask with , and aspirate off
[ice cold 1X PBS]
?. [Dissociate Adherent Cells]
Trypsinize cells:Add to flask an incubate at  for
[TrypLE]
37 °C
?. [Dissociate Adherent Cells]
Spin down nuclei solution at  at .
4 °C
?. [Dissociate Adherent Cells]
Resuspend pellet in  NIB:HEPES.Let nuclei incubate for
1 mL
on ice
?. [Dissociate Adherent Cells]
Repeat the spin down and resuspension (Repeat step 6.4-6.5)  for a second wash.
?. [Dissociate Adherent Cells]
Resuspend cells in   and hold on ice.
[NIB:HEPES]
?. [Quantify Nuclei Concentration]
Take aliquot of sample and dilute (10uL of nuclei suspension in  for a 1:10 dilution).
[NIB:HEPES]
?. [Quantify Nuclei Concentration]
Determine nuclei/uL concentration of diluted aliquot through Hemocytometer or BioRad TC20 Automated Cell Counter using manufacturer's recommended protocols. Adjust calculated concentration for the stock nuclei suspension.
?. [Quantify Nuclei Concentration]
Separate nuclei into 1 million nuclei aliquots and transfer into 5mL tubes. Add volume of NIB:HEPES to suspend 1M nuclei aliquots in .
5 mL
We generally generate multiple replicates of the 1M nuclei aliquots. We have also done up to 3M nuclei aliquots without any change to protocol which performed the same.
?. [Nuclei Fixation]
To the 5mL nuclei suspension, add   from a freshly opened ampule.
[16% formaldehyde]
?. [Nuclei Fixation]
Incubate nuclei at   for  on an orbital shaker set to  .
0 Room temperature
Centrifuge: 50 33
?. [Nuclei Fixation]
Centrifuge nuclei at  to pellet nuclei. Aspirate off solution, being careful not to disturb the pellet.
Centrifuge: 500 34, 5 min, 4 10
?. [Nuclei Fixation]
Wash pellet with  NIB:Tris.Centrifuge nuclei at  to pellet nuclei. Aspirate off solution, being careful not to disturb the pellet.
1 mL
Centrifuge: 500 34, 5 min, 4 10
?. [Nuclei Fixation]
Wash pellet with   1X NEBuffer 2.1 (ice cold)Transfer resuspended nucleito 1.5mL tube for improved pelleting.Centrifuge nuclei at  to pellet nuclei. Aspirate off solution, being careful not to disturb the pellet.
200 µl
Centrifuge: 500 34, 5 min, 4 10
?. [Nucleosome Depletion]
Resuspend pellet in  1X NEBuffer 2.1
760 µl
?. [Nucleosome Depletion]
Add  of 1% SDS and incubate at   on a ThermoMixer
40 µl
Centrifuge: 300 33, 20 min, 37 10
?. [Nucleosome Depletion]
Centrifuge nuclei at  to pellet nuclei. Aspirate off solution, being careful not to disturb the pellet.
Centrifuge: 500 34, 5 min, 4 10
?. [Nucleosome Depletion]
Resuspend pellet in  NIB:Tris. If there are multiple replicates they can be pooled at this point.
50 µl
If there is significant clumping, nuclei can bed loosed by a 10 min incubation in NIB:Tris and then triturated. If this does not work, loose strokes (B) on a douce homogenizer works as well.
?. [Nucleosome Depletion]
Take aliquot of sample and dilute (5uL of nuclei suspension in  for a 1:10 dilution).
[NIB:Tris]
?. [Nucleosome Depletion]
Determine nuclei/uL concentration of diluted aliquot through Hemocytometer or BioRad TC20 Automated Cell Counter using manufacturer's recommended protocols. Adjust calculated concentration for the stock nuclei suspension.
?. [Restriction Digest and Proximity Ligation]
For restriction digest, use a promiscuous cutter. We have used AluI which is a blunt end 4-cutter.Combined pooled nuclei into a single tube.
?. [Tagmentation]
Prepare tagmentation plateCombine with  per 96 well iTSM plate.Pipette out 8uL per well of TD Buffer: Nuclei solution.Add 1uL of 2.5uM uniquely indexed SBS12-U-ME TSM per well.Final reagents per well in tagmentation plate.   AB1ComponentVolume per well (9uL total)2~20ng DNA3.5 ul (~1750 nuclei at a concentration of 500 nuclei/uL)3iTSM1 uL 2.5uM SBS12-U-ME TSM4TD Buffer4.5ul 2X TD Buffer
[500 nuc/uL solution]
[2X TD Buffer]
AB1ComponentVolume per well (9uL total)2~20ng DNA3.5 ul (~1750 nuclei at a concentration of 500 nuclei/uL)3iTSM1 uL 2.5uM SBS12-U-ME TSM4TD Buffer4.5ul 2X TD Buffer
Hold SBS12-U-ME TSM plate on ice while pipetting out. Also hold nuclei and tagmentation plate on ice until plate is fully prepared.
?. [Tagmentation]
Incubate at  for  while shaking at  .
55 °C
Centrifuge: 300 33
Shaking helps keep nuclei suspended during tagmentation.
?. [Tagmentation]
Place plate on ice following incubation and hold for  to allow reactions to drop temperature.
?. [Tagmentation]
Pool the full plate while maintaining plate and pool on ice. Add  to pool. Hold pool on ice while preparing the PCR plate. Nuclei are stable on ice for several hours.
[5mg/mL DAPI]
If there are any visible chunks in pool, strain again through 40um cell strainer.
?. [Single-cell sorting]
Prepare a 96 well plate with  per well , diluted with ddH2O.
[1X TD Buffer]
?. [Single-cell sorting]
From the DAPI stained pool, sort 15 nuclei per well mimicking the above nuclei gating strategy. If possible, hold the sample chamber and pool at  in your sorter settings.
4 °C
The number of nuclei per well for PCR is determined by the number of uniquely indexed transposases going into tagmentation. For 96 reactions (a single iTSM plate), it has been emperically deteremined to be 15 nuclei per well based on a 5% barnyard collision rate.
?. [Single-cell sorting]
Following sorting into the 96 well plate, centrifuge the plate at   to ensure nuclei are within the solution.SAFE STOPPING POINT: Plates can be frozen (stored at )after spin down for at least 1 week without measurable decrease in library quality.
Centrifuge: 500 34, 5 min, 4 10
-20 °C
?. [Gap fill and adapter switching]
Add  to each well.
[0.1% SDS]
?. [Gap fill and adapter switching]
Add  to each well.
[NPM]
It is crucial to dd 0.1% SDS and NPM in step-wise fashion and not pre-combined. High SDS concentration will disrupt polymerases in NPM.
?. [Gap fill and adapter switching]
Gap-fill molecules by incubating plate at  for  .   After extension, hold plate  to slow reaction.
72 °C
on ice
?. [Gap fill and adapter switching]
Add  to a final concentration of
[A14-LNA_ME at 1uM]
?. [Gap fill and adapter switching]
Use multiple rounds of linear extension to switch adapter on one side of the molecules.  for   for   for   for Hold at
98 °C
98 °C
59 °C
72 °C
10 °C
?. [Gap fill and adapter switching]
Add .SAFE STOPPING POINT: Plates can be frozen (stored at )after spin down for at least 10 weeks without measurable decrease in library quality.
[1% (v/v) Triton X-100]
-20 °C
?. [PCR]
Combine the following reagents for a RT-PCR reaction directly to the 96 well plate contained the samples. ddH2O, Q5U Master Mix and 100X SYBR can be made as a mastermix prior to added to wells.  AB1ReagentVolume per well2  Sample  16.5 ul3PCR primers: S70X (TruSeq i7) 10uM2.5 uL4PCR primers: N50X (Nextera i5) 10uM2.5 uL5ddH2O  3 uL  6Q5U 2X Master Mix25 ul7  100X SYBR    0.5uL  8Total50 ul for PCR
AB1ReagentVolume per well2  Sample  16.5 ul3PCR primers: S70X (TruSeq i7) 10uM2.5 uL4PCR primers: N50X (Nextera i5) 10uM2.5 uL5ddH2O  3 uL  6Q5U 2X Master Mix25 ul7  100X SYBR    0.5uL  8Total50 ul for PCR
?. [PCR]
Perform a real-time PCR , measuring SYBR fluorecence every cycle.  for   for   for   for Measure SYBR fluoresence.   for   Go to step 2 16 more times for 17 cycles total.  for Hold at  for Hold at  SAFE STOPPING POINT: Plates can be frozen (stored at )after spin down for at least 10 weeks without measurable decrease in library quality. Can also be held at  for at least 5 weeks.
98 °C
98 °C
55 °C
72 °C
72 °C
72 °C
10 °C
PCR takes between 15-17 cycles. Use SYBR fluoresence to guide when to pull libraries. Once fluoresence stops exponential growth, the libraries are ready to hold at 72C and then remove.
-20 °C
4 °C
?. [Library clean up]
Pool post-PCR ProductPool 25 uL from each well into 15mL conical tube.
?. [Library clean up]
Concentrate DNA via column clean upRun full pool volume through Qiaquick PCR purification column following manufacturer's protocol. Elute in .
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[10 mM Tris-HCl pH 8.0]
?. [Library clean up]
Clean by size selection with SPRI beadsPerform a 1X SPRI bead size selection (selecting for DNA > 200 bp).
?. [Library clean up]
Add   to column elution, once beads are at room temperature.Let mixture incubate at room temperature for .
[MagPure NGS Binding Beads]
?. [Library clean up]
Place tube on magnetic rack and wait for magnetic beads to pellet and elution to fully clear (roughly  ).Remove full volume of elution without disrupting bead pellet. Discard elution.
?. [Library clean up]
Resuspend bead pellet in freshly prepared .Remove full volume of elution without disrupting bead pellet. Discard ethanol.
[80% ethanol (v/v)]
?. [Library clean up]
Resuspend bead pellet in freshly prepared .Remove full volume of elution without disrupting bead pellet. Discard ethanol.
[80% ethanol (v/v)]
?. [Library clean up]
Remove tube from magnetic rack and spin briefly on tapletop centrifuge.Return to magnetic rack and once beads are pelleted again, remove any remaining ethanol from bottom of tube.
?. [Library clean up]
Resuspend beads off magnetic rack in  .Let mixture incubate at room temperature for    for DNA to fully become suspended.Place tube on magnetic rack and wait for magnetic beads to pellet and elution to fully clear (roughly  ).Remove full volume of elution without disrupting bead pellet and move elution to clean tube.
[10 mM Tris-HCl pH 8.0]
?. [Library clean up]
Qubit DNA HS QuantificationQuantify DNA concentration with 1uL eluted sample on Qubit DNA High-sensitivity kit following manufacturer's protocol.
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?. [Library clean up]
Agilent Tapestation 4150 QuantificationDilute sample to 2 ng/uL based on read out of Qubit by addition of 10mM Tris-HCl pH 8.0.Run 2 uL sample on Agilent Tapestation 4150 DNA High Sensitivity D5000 or D1000 following manufacturer's protocol.
?. [Sequencing]
Use Tapestation software to quantify DNA from 200-1000bp size.Dilute library to 1nM and prepare for sequencing following Illumina protocols.
?. [Sequencing]
Load libraries according to standard loading concentrations suggested by Illumina with the following cycle counts per read. Sequencing is standard chemistry.  ABCDE1ReadRead 1Read 2Index 1Index 22Number of Cycles747410103Custom Index Primer?NoNoNoNo
ABCDE1ReadRead 1Read 2Index 1Index 22Number of Cycles747410103Custom Index Primer?NoNoNoNo
?. [Restriction Digest and Proximity Ligation]
Centrifuge nuclei at  to pellet nuclei. Aspirate off solution, being careful not to disturb the pellet.
Centrifuge: 500 34, 5 min, 4 10
?. [Restriction Digest and Proximity Ligation]
Resuspend pellet in
[1X Cutsmart Buffer]
?. [Restriction Digest and Proximity Ligation]
Add
[10 U/uL AluI Enzyme]
?. [Restriction Digest and Proximity Ligation]
Incubate at   on a ThermoMixer
Centrifuge: 300 33, 120 min, 37 10
?. [Restriction Digest and Proximity Ligation]
Centrifuge nuclei at  to pellet nuclei. Aspirate off solution, being careful not to disturb the pellet.
Centrifuge: 500 34, 5 min, 4 10
?. [Restriction Digest and Proximity Ligation]
For proximity ligationResuspend nuclei in 100uL of the following mixture:  AB1ReagentVolume for 100uL mixture210X T4 DNA Ligase Buffer + ATP10 uL3ddH2O88 uL40.1 % TritonX-1001 uL (final concentration 0.01%)50.1 M DTT0.5 uL (final concentration 0.5mM)6T4 DNA  (400 U/uL)0.5 uL (200 U total)
AB1ReagentVolume for 100uL mixture210X T4 DNA Ligase Buffer + ATP10 uL3ddH2O88 uL40.1 % TritonX-1001 uL (final concentration 0.01%)50.1 M DTT0.5 uL (final concentration 0.5mM)6T4 DNA  (400 U/uL)0.5 uL (200 U total)
?. [Restriction Digest and Proximity Ligation]
Incubate reaction at
16 °C
[(12-16 hours)]
?. [Restriction Digest and Proximity Ligation]
Centrifuge nuclei at  to pellet nuclei. Aspirate off solution, being careful not to disturb the pellet.
Centrifuge: 500 34, 5 min, 4 10
?. [Restriction Digest and Proximity Ligation]
Resuspend pellet in
[NIB:HEPES]
?. [Restriction Digest and Proximity Ligation]
Take aliquot of sample and dilute (5uL of nuclei suspension in  for a 1:10 dilution).
[NIB:HEPES]
?. [Restriction Digest and Proximity Ligation]
Determine nuclei/uL concentration of diluted aliquot through Hemocytometer or BioRad TC20 Automated Cell Counter using manufacturer's recommended protocols. Adjust calculated concentration for the stock nuclei suspension.
?. [Restriction Digest and Proximity Ligation]
Dilute nuclei to 500 nuclei/uL with NIB:HEPES.