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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for the s3 ATAC dhe. s3ATAC is a simplified sci-ATAC protocol using in vitro tagmentation to capture regions of open chromatin genome-wide in single cells. </div><div class = "text-block"><span>For more information see </span><a href="https://mulqueenr.github.io/scatac" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://mulqueenr.github.io/scatac/</span></a><span style = ":;"> </span></div></div>

[STEPS]
?. [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.
?. [Dissociate Tissue]
Different isolation techniques are h ighlighted across the s3 protocol family and can be used interchangeably for downstream processing. For s3ATAC, we used primary human and mouse flash frozen brain samples.
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 Tissue]
Obtain a flash frozen primary tissue sample and store in either -80C or on dry ice.
?. [Dissociate Tissue]
Prechill instruments for dissection of tissue.Chill petri dish over dry ice to act as surgery stage.Chill razors by placing the blades in dry ice.Fill 7mL capacity dounce homogenizer with   NIB:HEPES solution.Chill dounce homogenizer vessel with solution in wet ice. Chill dounce homogenizer pestles by holding in 70% ethanol in 15mL tubes on ice.      Fill 7mL capacity dounce homogenizer with  NIB:HEPES solution.Chill dounce homogenizer vessel with solution in wet ice. Chill dounce homogenizer pestles by holding in 70% ethanol in 15mL tubes on ice.      Immediately prior to using dounce pestles, rinse with chilled diH2O, wipe with kim wipes to remove most of the ethanol.
2 mL
?. [Dissociate Tissue]
Section primary tissue:
No need to mince any chunks at this point, just keep as frozen as possible.
Only need a small fraction of most samples (~5mg)
?. [Dissociate Tissue]
Transfer tissue chunk via razor blade to NIB:HEPES-filled and chilled dounce vessel.Give  to equilibrate to salt concentration of NIB:HEPES buffer prior to douncing.
?. [Dissociate Tissue]
Dounce homogenize:Homogenize tissue with 5 loose (A) pestle strokes. Wait for   to cells to equilibrateDounce with tight pestle (B) ~10 more times.Go until solution is a slush, persisting chunks are fine.Wait for  to cells to equilibrateDounce with tight pestle (B) ~10 more times.Go until solution is a slush, persisting chunks are fine.
Go as slowly as possible, do not surpass the water-level, do not introduce bubbles. Not all chunks have to be dounced fully.
?. [Dissociate Tissue]
Strain cells:Any persisting large debris will fall to the bottom of the tube after  .Take the supernatant to pass through a 35uM strainer first. Following the supernatants passage, take remaining dounced solution (and persisting debris) and attempt to pass through the strainer as well. This is done to maximize cell passage through the strainer prior to clogging.
Multiple strainers may be necessary.
Transfer liquid across strainers until most is gone
?. [Dissociate Tissue]
Pool strained solution into 15mL tube.
?. [Dissociate Tissue]
Resuspend pellet in  NIB:HEPES.
1 mL
?. [Dissociate Tissue]
Repeat the spin down and resuspension (Repeat step 3.8-9)  for a second wash.
?. [Quantify Nuclei Concentration]
Take aliquot of sample and dilute (10uL of nuclei suspension in  for a 1:10 dilution).
[NIB:HEPES]
?. [Dissociate Tissue]
Spin down nuclei solution at  for  at .
4 °C
?. [Dissociate Tissue]
Resuspend cells in   and hold on ice.
[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]
Concentrate via centrifugation and buffer removal, or dilute via addition of NIB:HEPES to attain a nuclei solution of  and hold on ice until ready to proceed.
?. [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 (~5k nuclei at a concentration of 1.4K nuclei/uL)3iTSM1 uL 2.5uM SBS12-U-ME TSM4TD Buffer4.5ul 2X TD Buffer
[1400 nuc/uL solution]
[2X TD Buffer]
AB1ComponentVolume per well (9uL total)2~20ng DNA3.5 ul (~5k nuclei at a concentration of 1.4K 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 17 more times for 18 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 16-18 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
?. [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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