HDMA Raw Data Metadata

Greenleaf Lab SHARE-seq Protocol 1 of 15 Supplementary Note 1: SHARE-seq protocol General notes ● Before starting the experiment, clean materials with RNase Away and 70% ethanol, use autoclaved tips and tubes. Maintain samples and solutions ice-cold during the whole experiment if not specified otherwise. ● Default spin for cells at 500xg for 5 min w/ swing bucket centrifuge. ● Start with 100k-1M cells for fixation (more the better but ensure enough combinatorial space for final sublibraries) o Expect ~20% of original starting number of cells by library prep stage. ● Resuspend oligos to appropriate concentrations in IDTE buffer: o 1mM = linker oligos o 100µM = RT primer, TSO, barcoded oligos ● Add all RNase inhibitors fresh to mastermix. ● Enzymatics RI is the only RNase Inhibitor that seems to be compatible with Tn5 activity, so we switch cells from the PBS-2RI into PBS-RI buffer before ATAC. ● Some washing steps specifies keeping pellet/beads stable and adding buffer directly without disturbing the pellet/beads. This does affect final recovery. ● When removing supernatant, use a two-step removal method: first remove the majority of supernatant with P1000, and then remove the remaining amount with P200. It may help to tilt the 2mL tube completely horizontally as you get close to removing all the liquid to avoid removing the pellet while removing as much liquid as possible. ● 2mL lo-bind Eppendorf tubes has better cell/nuclei recovery than 1.5mL tubes only when centrifugation is performed in a swing bucket because it’s easier to completely aspirate the supernatant from 2mL tubes without disturbing the pellet at the tip of the 2mL tube. ● We split the bulk ATAC and RT reaction per sample into smaller tubes because the reaction efficiency is better with a smaller volume (even under the same concentration of reaction components). ● Where possible, check cell/nuclei quality and count using Countess at every major steps (ideally: pre-fix, preATAC, pre-splitpool, post-ligation. If necessary, can get away without counting at pre-fix). ● Check before every run to ensure sufficient reagents in stock. Greenleaf Lab SHARE-seq Protocol 2 of 15 Stock Buffer recipes: These buffers can be made in big batch ahead of time and stored for a few months Nuclei Isolation Buffer (NIB) (Keep at 4˚C) Stock solution Final conc. Vol to add (mL) 1M Tris HCl pH 7.5 10mM 0.5 5M NaCl 10mM 0.1 1M MgCl2 3mM 0.15 10% NP40 (IGEPAL CA-630) 0.1% 0.5 Ultrapure water 48.75 Total 50 2x RCB (Keep at room temp) Stock solution Final conc. Vol to add (mL) 1M Tris HCl pH 8.0 100mM 1 5M NaCl 100mM 0.2 20% SDS 0.4% 0.2 Ultrapure water 8.58 Total 10 2x BW (Keep at 4˚C) Stock solution Final conc. Vol to add (mL) 1M Tris HCl pH 8.0 10mM 0.5 5M NaCl 2M 20 0.5M EDTA 1mM 0.1 Ultrapure water 29.4 Total 50 1x B&W-T (Keep at 4˚C) Stock solution Final conc. Vol to add (mL) 1M Tris HCl pH 8.0 5mM 0.25 5M NaCl 1M 10 0.5M EDTA 0.4% 0.05 10% Tween 20 0.05% 0.25 Ultrapure water 39.45 Total 50 IDTE (oligo resuspension) (Keep at room temp) Stock solution Final conc. Vol to add (mL) 1M Tris HCl pH 8.0 10mM 0.5 0.5M EDTA 0.1mM 0.01 Ultrapure water 49.5 Total 50 Greenleaf Lab SHARE-seq Protocol 3 of 15 STE (Keep at room temp) Stock solution Final conc. Vol to add (mL) 1M Tris HCl pH 8.0 10mM 0.5 5M NaCl 50mM 0.5 0.5M EDTA 1mM 0.1 Ultrapure water 48.9 Total 50 2x TD buffer (Keep at -20C) Stock solution Final conc. Vol to add (mL) 1 M Tris-HCl pH 7.6 20 mM 2 mL 1 M MgCl2 10 mM 1 mL *If preparing Tris from solid base, adjust pH to 7.6 before the addition of DMF Dimethyl Formamide (DMF) 20% 20 mL Ultrapure water NA Bring up to 100 mL Total 100 mL Greenleaf Lab SHARE-seq Protocol 4 of 15 Split pool oligo plate preparation: Can be made in big batch ahead of time and stored at -20C for a few months 1. Thaw Round1, 2, 3 barcode oligo plates at room temp and let completely thaw (few hours or also try water bath). a. Very critical step that all oligos are at room temp before use. b. Spin the plates down using a plate adapter on a swing bucket or plate-compatible centrifuge. 2. Prepare each linker oligo dilution in tubes as below: a. 120µL Round 1 linker oligo (1mM) + 11,880µL STE buffer b. 120µL Round 2 linker oligo (1mM) + 9,480µL STE buffer c. 144µL Round 3 linker oligo (1mM) + 9,360µL STE buffer 3. Dispense following amounts of linker oligos into each 96 well PCR plate: a. 90µL diluted Round 1 linker oligo b. 88µL diluted Round 2 linker oligo c. 86µL diluted Round 3 linker oligo 4. Use a multichannel pipette to transfer the barcode oligo plates to the appropriate Round linker plate: a. 10µL Round 1 barcode oligos b. 12µL Round 2 barcode oligos c. 14µL Round 3 barcode oligos 5. Seal plates with aluminum adhesive cover and ensure that each well is well protected. Set up thermocycler as below: Temp Time 95˚C 2:00 min -1˚C/1 min 20˚C 2:00 min 4˚C Hold Total ~1h 26min 6. Check each well to make sure that there was not significant evaporation. If so, add appropriate amount of water to compensate. 7. Aliquot 10µL of the annealed oligos to a new plate (should be enough for 9x plates for each round). Use a liquidator or a liquid handling platform for distribution. Seal carefully to ensure proper seal at each well with aluminum foil seal and store at -20˚C. Greenleaf Lab SHARE-seq Protocol 5 of 15 Buffer preparation Prepare following buffers fresh on the day of experiment, keep on ice unless otherwise specified. Recommended order to prepare buffers: PBS-2RI, 2x TB Omni, 1x TB Omni, PBS-RI, NIB-RI. Scale according to the number of samples and number of reactions needed PBS-2RI Vol for 1 sample (µL) 1xPBS 3000 7.5% BSA 16.05 Enzymatics RI 7.5 SUPERase RI 3.75 Total 3027.3 PBS-RI Vol for 1 rxn (µL) 1x PBS 5 Enzymatics RI 0.0125 Total 5.0125 NIB-RI Vol for 1 sample (µL) NIB 8323 Enzymatics RI 21 SUPERase RI 21 Total 8365 *6.565mL fixed volume of NIB-RI required given round 1 barcoding uses a single 96 well plate, another 1.8mL per sample required 2x TB (Omni) Vol for 1 rxn (µL) 0.2M Tris-acetate 8.25 5M K-acetate 0.66 1M Mg-acetate 0.5 10% Tween-20 0.5 1% Digitonin 0.5 100% DMF 8 Ultrapure water 6.59 Total 25 Greenleaf Lab SHARE-seq Protocol 6 ATAC (~20min) * Includes time-sensitive steps, make sure the appropriate buffers are ready ahead of time. * This protocol assumes nuclei isolation, permeabilization, and fixation has already occurred during tissue dissociation. 1. Set swing bucket centrifuge to 4C. 2. Prepare 1xTB: 1x TB Omni Vol for 1 rxn (µL) 2x TB (Omni) 25 Ultrapure water 16.45 PIC 0.2 Enzymatics RI 0.85 Total 42.5 3. After appropriate nuclei dissociation protocol and fixation, mix the desired amount of cells with 500µL of PBS-RI and mix thoroughly, especially if it contains iodixanol. 4. Spin down at 500xg for 5 min at 4C in swing bucket centrifuge. 5. Prepare bulk transposition as follows, each reaction is ~40k cells. Prepare just enough based on the total number of ATAC reactions needed. ATAC rxn Vol for 1 rxn (µL) Sample resuspended in PBS-RI 5 1xTB (Omni) 42.5 SHARE-ATAC Tn5 WJG Mar 2023 2.5 Total 50 a. Resuspend each sample pellet in PBS-RI in appropriate volumes (5 µL per ATAC reaction). b. Assemble the ATAC reactions per sample with appropriate volume of 1xTB and Tn5. Tn5 is viscous and should be pipette mixed in slowly. c. Mix well and distribute 50µL per well to a lo-bind 96 well plate on a cooling block on ice (it’s ok if the last well gets <50µL). Keeping plate cold is essential to control the Tn5 activity before the reaction starts. Seal plate, and incubate at 37˚C for 30 min with shaking at 500 rpm. d. At this time, thaw the 5x RT buffer, RT primer (100µM), and dNTPs. 6. Pool ATAC reactions by sample in 2mL lo-bind tubes, keeping the plate and tubes on ice as much as possible. 7. Spin down at 500xg for 5 min at 4C, remove supernatant. 8. Wash pellet gently without resuspending with 1mL NIB-RI. 9. Spin down at 500xg for 5 min at 4C. 10. Remove supernatant and resuspend pellet thoroughly in appropriate volumes of EB per sample (10µL per RNA RT reaction). Greenleaf Lab SHARE-seq Protocol 7 RT (~45 min) 1. Prepare RT mix (optimized for 100k cells per 50µL rxn): RT mix Vol for 1 rxn (µL) 5x RT buffer 10 Ultrapure water 1.56 dNTPs 2.5 Enzymatics RI 0.31 SUPERase RI 0.63 RT primer (100µM) 5 50% PEG (wide bore tips) 15 Maxima H Minus RT (add right before rxn) 5 Total 40 2. Start the RT protocol on thermocycler – the protocol should have a 50˚C hold so that when the samples are put in the machine, the hold is released and the 10min at 50˚C can be started right away. 3. Add appropriate volumes of RT mix to cells in EB (40uL RT mix + 10uL sample per reaction). 4. Split RT mix into PCR strips with 50µL/tube. 5. Set up thermocycler as below: Temp Time 50˚C Hold 50˚C 10 min 8˚C 12s 3 cycles 15˚C 45s 20˚C 45s 30˚C 30s 42˚C 2 min 50˚C 3 min 50˚C 5 min Total 37 min 6. All steps below are at room temperature. 7. Thaw barcode oligos plates, block 1, 2, 3 oligos and T4 DNA ligase buffer at room temp. Place NIB-RI at room temp. Set centrifuge to room temp. 8. Add 300µL NIB-RI to a new 2ml lobind tube per sample and pool RT reactions into it, pipette mix well. 9. Spin down at 500xg for 5 min at room temp in swing bucket centrifuge. 10. Wash with 500µL NIB-RI without disturbing the pellet. 11. Spin down at 500xg for 5 min at room temp in swing bucket centrifuge. 12. Remove supernatant and resuspend pellet thoroughly with appropriate volumes of NIB-RI. The total volume for a single 96 well plate of hybridization is 1080µL NIB-RI. Divide by the number of independent samples. a. Count nuclei to confirm nuclei quality and cell count (pre-splitpool) Greenleaf Lab SHARE-seq Protocol 8 Hybridization-ligation (~5hrs) 1. Make sure that all oligo plates are thawed to room temp and spun down before hybridization. 2. Prepare hybridization buffer in 5mL tube: Hybridization buffer Vol for 96 wells (µL) Ultrapure water 2589.3 T4 ligase buffer (10x) 540 SUPERase RI 13.5 Enzymatics RI 43.2 10% NP-40 54 Total 3240 3. Add appropriate volumes of hybridization buffer to resuspended samples in NIB-RI, mix well. If there are multiple samples, then divide the hybridization buffer appropriately for number of sample. 4. Using a multichannel trough, aliquot 40µL sample mixture to Round 1 plate, mix, and seal plate. 5. Shake at 300rpm for 30min at 25oC. 6. Prepare blocking oligo 1 mixture: Blocking oligo 1 Vol (µL) Round 1 blocking (1mM) 25.3 T4 ligase buffer (10x) 211.2 Ultrapure water 915.5 Total 1152 a. Distribute 90µL each to a PCR strip (12x) and use a P20 multichannel to distribute blocking oligo and mix. 7. Using a whole new P10 pipette tip box, add 10µL blocking oligo 1 mixture to each well, mix, and seal plate. 8. Shake at 300 rpm for 30 min at 25oC. 9. Pool samples into a trough, and using a whole new P200 pipette tip box, aliquot 50µL of mixture to Round 2 plate. a. Use a P200 multichannel to aspirate and pool into a new basin and redistribute using a multichannel. 10. Mix, seal plate, and shake at 300rpm for 30min at 25oC. 11. Prepare blocking oligo 2 mixture: Blocking oligo 2 Vol (µL) Round 2 blocking (1mM) 30.4 T4 ligase buffer (10x) 211.2 Ultrapure water 910.4 Total 1152 a. Distribute 90µL each to a PCR strip (12x) and use a P20 multichannel to distribute blocking oligo and mix. 12. Using a whole new P10 pipette tip box, add 10µL blocking oligo 2 mixture to each well, mix, and seal plate. 13. Shake at 300rpm for 30min at 25oC. 14. Pool samples into a trough, using a whole new P200 pipette tip box, aliquot 60µL of mixture to Round 3 plate. a. Use a P200 multichannel to aspirate and pool into a new basin and redistribute using a multichannel. 15. Mix, seal plate, and shake at 300rpm for 30min at 25oC. 16. Prepare blocking oligo 3 mixture: Blocking oligo 3 Vol (µL) Round 3 blocking (1mM) 26.5 10% NP-40 (can be replaced with water) 11.5 Ultrapure water 1114 Total 1152 b. Distribute 90µL each to a PCR strip (12x) and use a P20 multichannel to distribute blocking oligo and mix. 17. Using a whole new P10 pipette tip box, add 10µL blocking oligo 3 mixture to each well. mix, and seal plate. 18. Shake at 300rpm for 30min at 25oC. 19. Pool samples to a trough and add 70µL 7.5% BSA to the solution and mix well. c. Can filter while transferring using a FlowMi 40µm filter to remove multiplets. Greenleaf Lab SHARE-seq Protocol 9 20. Transfer to 4x lo-bind 2mL tubes, spin down at 500xg for 5 min at room temp in a swing bucket centrifuge. 21. Remove supernatant and add gently 1mL NIB-RI to pellet without resuspending. 22. Spin down at 500xg for 5 min at room temp, remove supernatant. 23. Resuspend with 85µL NIB-RI: start with 40µL, pool all tubes, measure volume and top up to 85 µL. 24. Prepare Ligation mix: Ligation buffer Vol (µL) Ultrapure water 251.8 T4 ligase buffer (10x) 40 SUPERase RI 1 Enzymatics RI 3.2 10% NP-40 4 T4 ligase 20 Total 320 25. Mix 80µL sample to 320µL ligation mix. 26. Distribute 50µL to 8 PCR tubes. 27. Shake at 300rpm for 30 min at 25oC. 28. Pool samples, spin down at 500xg for 5 min at room temp. 29. Remove supernatant and wash pellet with 1mL NIB-RI. 30. Spin down at 500xg for 5 min at room temp. 31. Resuspend thoroughly (10x pipetting) with **200µL NIB-RI and count nuclei (post-ligation). d. Volume of final resuspension depends on expected nuclei recovery. Generally, can assume between 100-200k but will change depending on cell type and the number of cells at the start of split and pool. e. **400µL assumes 200k final recovery. Adjust appropriately for desired number of cells per library. Reverse crosslinking & pull down (2:45 hrs) 1. Based on desired sub-library size (# cells), dilute the cells appropriately for each 50µL reaction. a. Range: 2,000-20,000 cells / 50µL. b. Each sub-library is processed independently. c. N = number of sublibraries 2. Prepare reverse crosslinking master mix as follows. RC MM Vol for 1 sublib (µL) 2x RCB Buffer 50 proteinase K (20mg/mL) 2 SUPERase RI 1 Total 53 3. For each tube of 50µL sample, add 53 µL of RC MM. 4. Incubate at 55˚C for 1h. a. Do not incubate longer, decreases yield 5. Recommended stopping point, store product at -80˚C for few weeks 6. Thaw libraries, if necessary, PMSF/IPA, equilibrate B&W buffers to room temp. 7. After thaw, stop proteinase K by adding 5µL 100mM PMSF/IPA (don’t freeze thaw) and incubate at room temp for 10 min. 8. Prepare following buffers (N = number of sublibraries): 1x B&W-T/RI Vol for 1 sublib (µL) 1x B&W-T 400 SUPERase RI 4 Total 404