Reagent setup
Annealing buffer
Mix 10 μl 1 M Tris-HCl (pH 8.0), 10 μl 5 M NaCl, 2 μl 0.5 M EDTA (pH=8.0), 978 μl Nuclease-free Water for 1 ml Annealing buffer. Stored at -20°C for up to 1 year.
Coupling buffer
Mix 50 μl 1 M Tris-HCl (pH 7.5), 20 μl 5 M NaCl, 2 μl 50 mM EDTA (pH=8.0), 10 μl 10% TritonX-100, 1 μl 1 M DTT, 500 μl glycerol, and 417 μl Nuclease-free Water for 1 ml coupling buffer. Stored at -20°C for up to 1 year.
1× DPBS-0.5%BSA-RI (0.2U/μl)
Mix 166.7 μl 30% (w/v) BSA, 50 μl RiboLock RNase Inhibitor (40 U/μl), and 10 ml 1× DPBS. The buffer can be stored at -20°C for up to 2 weeks.
0.5×DPBS-0.5%BSA-RI (0.2U/μl)
Mix 166.7 μl 30% (w/v) BSA, 50 μl RiboLock RNase Inhibitor (40 U/μl), 5 ml 1× DPBS, and 5 ml Nuclease-free Water. The buffer can be stored at -20°C for up to 2 weeks.
1.1× lysis buffer
Mix 220 μl 1 M Tris-HCl (pH8.0), 44 μl 5 M NaCl, 440 μl 10% SDS and 19.3 ml Nuclease-free Water for 20 ml 1.1× lysis buffer. Sterilize through 0.22 μm filter, store at room temperature (RT, 20–25 °C) for up to 1 month.
Omni-ATAC RSB
Mix 500 μl 1 M Tris-HCl (pH 7.5), 100 μl 5 M NaCl, 150 μl 1 M MgCl2, and 49.25 ml Nuclease-free Water for 50 ml omni-ATAC RSB. Sterilize through 0.22 μm filter, stored at 4°C for up to 2 months.
RSB-DTN
Mix 487.5 μl Omni-ATAC RSB, 5 μl 10% Tween-20, 5 μl 10% IGEPAL CA-630 and 2.5 μl 2% Digitonin. Make fresh.
RSB-T
Mix 10 ml Omni-ATAC RSB and 100 μl 10% Tween-20. Store at 4°C for up to 1 month.
0.1% digitonin
Mix 0.5 μl 2% digitonin stock with 9.5 μl Nuclease-free Water. CRITICAL Make fresh and avoid freeze thaw cycles. The Promega 2% digitonin stock is dissolved in DMSO. Thaw the digitonin stock at room temperature.
4× THS TD buffer
Mix 132 μl 1 M Tris-HCl (pH8.0), 52.8 μl 5 M Potassium acetate, 40 μl 1 M Magnesium acetate, 640 μl N, N-Dimethylformamide (DMF) and 135.2 μl Nuclease-free Water for 1 ml 4× THS TD buffer. Store at -20°C for up to 6 months.
2× Stop Buffer
Mix 500 μl 1x DPBS, 40 μl 0.5M EDTA (pH=8.0), 66 μl 30% BSA and 394 μl Nuclease-free Water for 1 ml 2× Stop Buffer. Make fresh.
50% (w/w) PEG8000
Mix 5 g PEG8000 and 5 ml Nuclease-free Water, sterilize through 0.22 μm filter, aliquot and store at 4°C for up to 2 months.
5× NaCl RT Buffer
Mix 1.25 ml Tris-HCl (pH =8.0), 375 μl 5 M NaCl, 75 μl MgCl2, 250 μl 1 M DTT, 3.05 ml Nuclease-free Water to prepare 5 ml 5× NaCl RT Buffer. Sterilize through 0.22 μm filter, aliquot and store at -20°C for up to 2 months.
80% (v/v) ethanol
Mix 4 ml absolute ethanol with 1 ml Nuclease-free Water. Make fresh.
Procedures
Prepare Tn5 transposome complex Timing 2.5 h
1. Dissolve ME_S5, ME_S7 and ME_bottom oligos respectively in annealing buffer to a final concentration of 100 μM.
2. Mix 25 μl ME_S5 oligo (100 μM) with 25 μl ME_Bottom oligo (100 μM) in a PCR tube, and anneal in a thermocycler as follows: 98 °C for 3 min, and slowly cool down to 16 °C with a temperature ramp of −0.1 °C/s, to generate S5_adaptor (50 μM).
3. Similarly, mix 25 μl ME_S7 oligo (100 μM) with 25 μl ME_Bottom oligo (100 μM) in a PCR tube and anneal to form S7_adaptor (50 μM).
4. Dilute the annealed 50 μM S5_adaptor or S7_adaptor with Nuclease-free Water to 20 μM.
5. To assemble Tn5-S5/S7 transposome, add 12 μl S5_adaptor (20 μM), 12 μl S7_adaptor (20 μM), 48 μl purified Tn5 (0.5 μg/μl), and 88 μl coupling buffer to a 1.5 ml tube, mix thoroughly by gently pipetting up and down 10 times. Incubate at room temperature for 1 h, then store at -20 °C.
6. To assemble Tn5-S7/S7 transposome, add 24 μl S7_adaptor (20 μM), 48 μl purified Tn5 (0.5 μg/μl), and 88 μl coupling buffer to a 1.5 ml tube, mix thoroughly by gently pipetting up and down 10 times. Incubate at room temperature for 1 h, then store at -20 °C.
PAUSE POINT The assembled Tn5 transposome could be stored at -20°C for up to 6 months.
Prepare ISSAAC-seq lysis plate Timing 30 min
CRITICAL Be careful to avoid the solution splashing when handling plates, which may cause index contamination among different wells. Multiple plates can be prepared at the same time for convenience.
7. Take out the “scATAC 10 µM i7 index plate” (refer to our plate-based scATAC-seq workflow, Nat Protoc, PMID: 34282334) and thaw at room temperature.
8. Mix 9 volumes of 1.1× Lysis Buffer and 1 volume of Truseq_S5_short primer (100 µM in Nuclease-free Water) to reach a 10 µM concentration in 1× Lysis Buffer. Aliquot 1 µl to each well of a new 384-well plate. Label the plate as “ISSAAC lysis plate” and with the date.
9. Briefly centrifuge the thawed “scATAC-seq 10 µM i7 index plate” from above. Transfer 1 µl of 10 µM i7 index to the ISSAAC lysis plate using a 16-channel multichannel pipette.
10. Briefly centrifuge the ISSAAC lysis plate and seal the plate. Leave the plate on ice and carry on the experiment until the cells are ready. Alternatively, store the plate at -80 °C.
CRITICAL Evaporation can still happen even in the -80 °C freezer due to occasional open and closing of the freezer door. Therefore, always check the liquid volume in the wells before carrying on experiments.
PAUSE POINT The sealed plates can be stored at −80 °C for up to 1 year.
Prepare nuclei from cell lines Timing 1 h
CRITICAL Work quickly and always keep the samples on ice unless otherwise indicated.
CRITICAL All centrifugation should be performed at 4 °C in a swing bucket centrifuge. DO NOT use fixed-angle centrifuges, which will cause severe cell loss during washes.
CRITICAL When removing the supernatant, be careful not to disturb the cell pellet.
11. Coat the tube: add 0.5 ml 1× DPBS–0.5%BSA-RI (0.2 U/μl) to a 1.5 mL tube, invert the tube three to five times, then aspirate all liquid in the tube and inside the cap and discard.
CRITICAL Cells may attach to the inner surface of the centrifugation tubes. Coating the tubes with 0.5% (w/v) BSA helps reduce cell loss when working with low amounts of cells.
12. Count cells using a C-Chip disposable hemocytometer and use Trypan Blue to check cell viability. Make sure more than 90% cells are viable.
13. Spin down 100,000 cells per sample, centrifuge at 500g, 4°C for 5 min.
CRITICAL Perform all centrifugations on a swing bucket centrifuge to reduce cell loss.
14. Wash the pellet once with 500 µl ice-cold DPBS-0.5% BSA-RI, centrifuge at 500g, 4°C, 5 min.
15. Resuspend cells in 50 µl ice-cold RSB-DTN- RI (0.8 U/μl) and leave on ice for 3 min.
16. Add 1 ml ice-cold RSB-T to the tubes (1.05 ml in total), invert 3 times to mix.
17. Centrifuge at 1000g, 4 °C for 8 min to collect the nuclei.
18. Aspirate all supernatants and hold on ice.
Chromatin tagmentation Timing 1 h
19. Prepare 50 μl chromatin tagmentation reaction for each sample：
Reagent Stock Final Volume (50 μl)
4×THS TD buffer 4× 1× 12.5 μl
0.1% Digitonin 0.1% 0.01% 5 μl
Tn5-S5/S7 - - 2.5 μl
RiboLock RNase inhibitor 40 U/μl 1.2 U/μl 1.5 μl
SUPERaseIn RNase inhibitor 20 U/μl 0.4 U/μl 1 μl
RnaseOUT RNase inhibitor 40 U/μl 0.8 U/μl 1 μl
Nuclease-free Water - - 26.5 μl
CRITICAL Tn5 should be the last component to add, i.e., add it right before use.
20. Resuspend each pellet in 50 μl tagmentation mixture by gently pipetting up and down 30 times.
CRITICAL Perform gentle and slow pipetting to avoid disrupting the nucleus structure. Make sure that the nuclei are intact after pipetting. Mix gently and avoid bubbles.
21. Incubate at 30°C, 800 rpm for 30 min.
22. Add 50 μl 2× Stop buffer to each sample and gently pipette up and down 6~8 times to mix.
23. Centrifuge at 1000g, 4 °C for 5 min. Remove the supernatants and wash the pellet twice with 200 μl ice-cold 0.5×DPBS-0.5%BSA-RI.
24. Carefully remove all supernatants and hold on ice.
CRITICAL The nuclei pellet may be invisible, be careful not to disrupt the nuclei at the bottom of the tube when aspirating the supernatant. It is okay to have 2–3 μl buffer leftover at this step.
Reverse transcription Timing 1.5 h
25. Prepare 100 μl RT reaction for each sample, slowly pipette up and down 10~20 times to mix.
Reagent Stock Final Volume (100 μl)
TruseqR1_oligo_dT 10 μM 2 μM 20 μl
dNTP 10 mM each 0.5 mM each 5 μl
Maxima H minus Reverse Transcriptase 200 U/μl 10 U/μl 5 μl
RiboLock RNase Inhibitor 40 U/μl 2 U/μl 5 μl
SUPERaseIn RNase Inhibitor 20 U/μl 0.2 U/μl 1 μl
RnaseOUT RNase Inhibitor 40 U/μl 0.4 U/μl 1 μl
5× NaCl RT Buffer 5× 1× 20 μl
50% PEG8000 50% 12% 24 μl
Nuclease-free Water - - 19 μl
CRITICAL The 50% PEG8000 solution is viscous. Pipette slowly to ensure that no liquid remains along the tube sidewalls and tips.
26. Resuspend each pellet with 100 μl RT buffer by gently pipetting up and down 50 times.
CRITICAL Perform gentle and slow pipetting to avoid disrupting the nucleus structure. Make sure that the nuclei are intact after pipetting. Mix gently and avoid bubbles.
27. Transfer the 100 μl RT mixture to a new PCR tube and incubated in a thermocycler for 10 min at 50˚C before cycling for three times at 8˚C for 12s, 15˚C for 45s, 20˚C for 45s, 30˚C for 30s, 42˚C for 2 min, and 50˚C for 3 min, followed by a final step at 50˚C for 5 min.
28. Transfer the RT mixture to a new, BSA coated and chilled 1.5 ml tube.
29. Centrifuge at 1000g, 4 °C for 5 min. Remove the supernatants and wash the pellet twice with 200 μl ice-cold 0.5×DPBS-0.5%BSA.
30. Carefully remove all supernatants and hold on ice.
CRITICAL After reverse transcription, the nuclei will be partially lysed and the number of intact nuclei will decrease. The pellet may be invisible, be careful not to disrupt the nuclei at the bottom of the tube when aspirating the supernatant. It is okay to have 2–3 μl buffer leftover at this step.
mRNA-cDNA hybrid tagmentation Timing 1 h
31. Set up RNA-DNA hybrid tagmentation mix, prepare 50 μl for each sample:
Reagent Stock Final Volume (50 μl)
4×THS TD buffer 4× 1× 12.5 μl
0.1% Digitonin 0.1% 0.01% 5 μl
Tn5-S7/S7 - - 1.5 μl
Nuclease-free Water - - 31 μl
32. Resuspend each pellet with 50 μl tagmentation buffer by gently pipetting up and down 30 times.
CRITICAL Perform gentle and slow pipetting to avoid disrupting the nucleus structure. Make sure that the nuclei are intact after pipetting. Mix gently and avoid bubbles.
33. Incubate at 37°C, 800rpm, 30 min.
34. Add 50 μl 2× Stop Buffer to each sample, mix thoroughly by gentle pipetting 6~8 times.
35. Centrifuge at 1000g, 4 °C for 5 min. Remove the supernatants and wash the pellet twice with 200 μl ice-cold 0.5×DPBS-0.5%BSA.
36. Carefully remove all supernatants and hold on ice.
CRITICAL The pellet may be invisible, be careful not to disrupt the nuclei at the bottom of the tube when aspirating the supernatant. It is okay to have 2–3 μl buffer leftover at this step.
EXO I digestion and Gap fill-in Timing 30 min
37. Prepare 50 μl reaction for each sample:
Reagent Stock Final Volume (50 μl)
dNTP 10 mM each 0.5 mM each 2.5 μl
Maxima H Minus Reverse Transcriptase 200 U/μl 8 U/μl 2 μl
Exo1 20 U/μl 2 U/μl 5 μl
5× NaCl RT Buffer 5× 1× 10 μl
Nuclease-free Water - - 30.5 μl
38. Resuspend each pellet in 50 μl reaction by gently pipetting up and down 30 times.
39. Incubate at 37°C for 15 min.
40. Centrifuge at 1000g, 4 °C for 5 min, aspirate all supernatants.
41. Add 600 μl ice-cold 0.5×DPBS-0.5%BSA to each sample, resuspend pellet and transfer to a FACS tube.
42. Stain with DAPI: Mix 1 μl DAPI stock solution (1 mg/ml; protect from light) with 9 μl ddH2O, and then add 1.5 μl of diluted DAPI solution to the 600 μl sample, vortex briefly and hold on ice.
FACS sorting Timing >30 min
43. Take out the “ISSAAC lysis plate” and thaw at room temperature. Briefly centrifuge to collect the liquid back to the bottom of each well.
44. Load the DAPI-stained sample and sort one DAPI positive nucleus to every well of the ISSAAC lysis plate except the well P24 which is left as a no cell control.
45. After sorting, seal the plate immediately, and centrifuge at 1,000 g for 1 min.
CRITICAL It is very important to centrifuge the plate to make sure the nuclei go down to the lysis buffer. Perform the centrifugation immediately after sorting every plate, which helps avoid losing the cells on the plate wall. (Refer to PMID: 34282334 for more suggestion on FACS optimization.)
PAUSE POINT After centrifugation, the plate containing the sorted nuclei can be stored at −80 °C for up to 3 months.
Pre-amplification and well barcodes addition Timing 1 h
46. Nuclei lysis: Incubate the plate at 65°C for 15 min, then centrifuge at 1000g for 1 min.
47. SDS quenching: carefully remove the sealing film, add 1 μl 10% Tween-20 to each well, then centrifuge the plate at 1000g for 1 min.
CRITICAL Be careful to avoid the solution splashing out of the wells.
CRITICAL The presence of SDS in the lysis buffer may inhibit the following PCR. The addition of excess Tween-20 neutralizes the negative effect of SDS on PCR.
48. Add 3 μl NEB Q5 High-Fidelity 2× Master Mix to each well. The total volume of PCR reaction in each well by now is 6 μl. Tightly seal the plate, then centrifuge at 1000g for 1 min.
49. Perform RNA library pre-PCR for individual cells in 384-well plate:
Step Temperature Time Number of cycles
Gap fill-in 72°C 5 min 1
Initial denaturation 98°C 1 min 1
Denaturation 98°C 20 sec 4
Annealing 63°C 20 sec
Extension 72°C 1 min
Hold 10°C ∞ 1
50. Centrifuge the plate at 1000g for 1 min.
51. Carefully remove the sealing film, add 1 μl NEB Q5 High-Fidelity 2× Master Mix and 1 μl Nextra_S5 short primer (10 μM in Nuclease-free Water) to each well. Tightly seal the plate, and then centrifuge at 1000 g for 1 min.
52. Perform ATAC+RNA library pre-PCR for individual cells in 384-well plate:
Step Temperature Time Number of cycles
Initial denaturation 98°C 1 min 1
Denaturation 98°C 20 sec 6
Annealing 63°C 20 sec
Extension 72°C 1 min
Hold 10°C ∞ 1
53. Centrifuge the plate at 1000 g for 1 min, then carefully remove the sealing film.
54. Put a Low Dead Volume Reservoir on top of the 384-well plate, invert and centrifuge at 1000g for 1 min to pool all wells from a plate into the reservoir.
55. Transfer the pre-amplified PCR products to a new 15 ml tube.
CRITICAL STEP Genomic fragments from each well is now barcoded by a unique i7 index. However, plate barcode has not been added yet at this stage. Different plates must be processed separately. DO NOT mix samples from different plates.
56. Purify the pre-amplified PCR products using Zymo DNA Clean &Concentrator kit and elute the products in 41 μl Nuclease-free Water. (Refer to PMID: 34282334 for demonstration of using an extender tube and a vacuum connector to purify large volume of reaction in a single column.)
Exonuclease I digestion and purification Timing 1 h
57. Prepare 50 μl EXO I digestion reaction for each sample to eliminate residual primers form pre-amplifications:
Reagent Stock Final Volume (50 μl)
10× NEB 3.1 buffer 10× 1× 5 μl
EXO I - - 5 μl
Purified pre-products - - 40 μl
58. Incubate at 37°C for 30 min.
59. Inactivate EXO I by heating at 80 °C for 2 min.
60. Purify the product by 1.2× VAHTS DNA Clean Beads, and elute in 16 μl Nuclease-free Water.
Final library amplification and plate barcodes addition Timing 1 h
61. Set up PCR reaction:
2.5 μl ATAC_plate_S5xx (10 μM)
5.0 μl Illumina P7 (10 μM)
2.5 μl RNA_plate_S5xx (10 μM)
25 μl Q5 High-Fidelity 2× Master Mix
15 μl Purified DNA from above
CRITICAL The ATAC_plate_S5xx and RNA_plate_S5xx primers have the ATAC/RNA sample barcodes. Use different primers for different samples if you want to sequence the samples in the same lane.
62. Perform PCR as follows:
Step Temperature Time Number of cycles
Initial denaturation 98°C 1 min 1
Denaturation 98°C 20 sec 8
Annealing 63°C 20 sec
Extension 72°C 1 min
Final extension 72°C 5 min
Hold 10°C ∞ 1
63. Resulting library was purified with 1.0× VAHTS DNA Clean Beads and elute in 20 μl Nuclease-free Water.
Library QC and sequencing Timing 4~6 d
64. Measure the concentration of each library by Qubit according to the manufacturer’s instructions.
65. Dilute the libraries to 1~10 ng/μl using Nuclease-free Water.
66. Check the library size distribution on an Agilent high-sensitivity chip. A broad peak with an average size of 200–1000 bp will be observed.
67. Sequence the libraries for 150 x 8 x 8 x 150 cycles on Novaseq 6000 platform (Illumina).