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
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
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.
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 2 months.
2× Stop Buffer
Mix 500 μl 1x PBS, 40 μl 0.5M EDTA (pH8.0), 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 -20°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.
1x DNB-0.5%BSA
Mix 50 μl 20x DNB (10x genomics), 16.7 μl 30% BSA and 933.3 μl Nuclease-free Water. Make fresh.
50% (v/v) glycerol
Mix 5 ml glycerol and 5 ml Nuclease-free Water. Sterilize through 0.22 μm filter, aliquot and store at 4°C for up to 3 months.
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-S5/S5 transposome, add 24 μl S5_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 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.
7. 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.
8. Count cells using a C-Chip disposable hemocytometer and use Trypan Blue to check cell viability. Make sure more than 90% cells are viable.
9. 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.
10. Wash the pellet once with 500 µl ice-cold DPBS-0.5% BSA-RI, centrifuge at 500g, 4°C, 5 min.
11. Resuspend cells in 50 µl ice-cold RSB-DTN- RI (0.8 U/μl) and leave on ice for 3 min.
12. Add 1 ml ice-cold RSB-T to the tubes (1.05 ml in total), invert 3 times to mix.
13. Centrifuge at 1000g, 4 °C for 8 min to collect the nuclei.
14. Aspirate all supernatants and hold on ice.
Chromatin tagmentation Timing 1 h
15. 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.
16. 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.
17. Incubate at 30°C, 800 rpm for 30 min.
18. Add 50 μl 2× Stop buffer to each sample and gently pipette up and down 6~8 times to mix.
19. Centrifuge at 1000g, 4 °C for 5 min. Remove the supernatants and wash the pellet twice with 200 μl ice-cold 0.5×PBS-0.5%BSA-RI.
20. 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
21. Prepare 100 μl RT reaction for each sample, slowly pipette up and down 10~20 times to mix.
Reagent Stock Final Volume (100 μl)
TruseqR2_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.
22. 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.
23. 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.
24. Transfer the RT mixture to a new, BSA coated and chilled 1.5 ml tube.
25. 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.
26. 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
27. 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-S5/S5 - - 1.5 μl
Nuclease-free Water - - 31 μl
28. 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.
29. Incubate at 37°C, 800rpm, 30 min.
30. During incubation, take out the 20X Nuclei Buffer, ATAC Buffer B, Reducing Agent B, and Barcoding Reagent B from -20 °C, and Single Cell ATAC Gel Beads v1.1 from -80 °C (10X genomics, Chromium Next GEM Single Cell ATAC Library Kit and Gel Bead Kit v1.1), equilibrate to room temperature.
CRITICAL Equilibrate to room temperature at least 30 min before loading the chip.
31. Add 50 μl 2× Stop Buffer to each sample, mix thoroughly by gentle pipetting 6~8 times.
32. 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.
33. 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
34. 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
35. Resuspend each pellet in 50 μl reaction by gently pipetting up and down 30 times.
36. Incubate at 37°C for 15 min.
37. Centrifuge at 1000g, 4 °C for 5 min, aspirate all supernatants.
38. Wash the pellet twice with 150 μl of ice-cold 1x DNB (10x genomics)-0.5%BSA. Centrifuged at 1000g 4 °C for 5 min.
39. Remove all supernatants and hold on ice.
GEM Generation and Barcoding Timing 2 h
CRITICAL The following steps are modified form “Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 User Guide” (CG000209, 10x genomics).
40. Resuspend cells in ~15 µl of 1× DNB-0.5%BSA. Mix 2 µl cell suspension with 8 µl 0.4% trypan blue and count the nuclei using a C-Chip disposable hemocytometer.
CRITICAL Set a proper resuspension volume based on the total cell number and targeted loading concentration.
41. Prepare “Transposed Nuclei”: Add 7 µl ATAC buffer B, (8-x) µl Qiagen buffer EB, and x µl nuclei (5000 ~ 10000 nuclei) in order, to a chilled 1.5 ml tube. The total volume is 15 µl. Pipette mix 10 times using a 10 µl tip. Centrifuge briefly and hold on ice.
42. Prepare Master Mix: Add 56.5 µl Barcoding Reagent B, 1.5 µl Reducing Agent B, and 2 µl Barcoding Enzyme in order, to a chilled 1.5 ml tube. Pipette mix ~ 10 times using a 10 µl tip. Hold on ice.
43. Assemble CHIP H following the 10x instructions.
44. Dispense 50% Glycerol into Unused Chip Wells.
45. Add 60 µl master mix to 15 µl transposed nuclei, Pipette mix 20 times. Using the same pipette tip, dispense 70 μl Master Mix + Transposed Nuclei into the bottom center of each well in row labeled 1 without introducing bubbles.
46. Load Gel beads and Partitioning oil to each well following the 10x instructions.
47. Attach 10x Gasket, run the chip in the controller immediately.
48. Transfer the 100 μl GEMs to a chilled PCR 8-tube strip following the 10x instructions.
49. GEM incubation: incubate the PCR strip in a thermal cycler with the following program.
Temperature Time Number of cycles
72°C 5 min 1
98°C 30 sec 1
98°C 10 sec 12
59°C 30 sec
72°C 1 min
15°C ∞ 1
PAUSE POINT The GEMs could be stored at 15°C for up to 18 h or at -20°C for up to a week.
Post GEM incubation Cleanup Timing 1 h
50. Follow “Step 3” in the Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 User Guide (CG000209), but elute the final products in 35.5 µl Elution solution 1 (step 3.2 j).
PAUSE POINT The eluted products could be stored at 4°C for up to 72 h or at -20°C for up to 2 weeks.
Library construction Timing 2 h
51. Set up pre-amplification reaction as follows:
5 μl ATAC_droplet_N7xx (10 μM)
5 μl Illumina P5 (10 μM)
5 μl RNA_droplet_N7xx (10 μM)
50 μl 10x Amp Mix
35 μl Purified DNA from above
52. Incubate in a thermal cycler with the following protocol for pre-amplification:
Step Temperature Time Number of cycles
Initial denaturation 98°C 1 min 1
Denaturation 98°C 20 sec 7
Annealing 63°C 20 sec
Extension 72°C 20 sec
Final extension 72°C 1 min 1
Hold 10°C ∞ 1
53. Final ATAC library amplification: Take 50 µl of the pre-PCR product as ATAC pre-library, and purify with 1.0x VAHTS DNA clean bead, then elute in 40.5 μl Nuclease-free Water.
PAUSE POINT The ATAC pre-library could be stored at 4°C for up to 72 h or at -20°C for up to 2 weeks.
54. Setup ATAC library amplification reaction as follows:
5 μl ATAC_droplet_N7xx (10 μM)
5 μl Illumina P5 (10 μM)
50 μl Q5 High-Fidelity 2× Master Mix
40 μl Purified DNA from above
CRITICAL The ATAC_droplet_N7xx primers have the ATAC library sample barcodes. Use the same ATAC_droplet_N7xx primer for the same sample in pre-amplification and final ATAC library amplification. Use different primers for different samples if you want to sequence the samples in the same lane.
55. Perform ATAC library PCR as follows:
Step Temperature Time Number of cycles
Initial denaturation 98°C 1 min 1
Denaturation 98°C 20 sec 7
Annealing 63°C 20 sec
Extension 72°C 20 sec
Final extension 72°C 1 min 1
Hold 10°C ∞ 1
56. Purify the final ATAC library using 1.0× VAHTS DNA cleaning beads and elute in 20 μl Nuclease-free Water.
57. Final RNA library amplification: Purify the remaining 50 µl pre-PCR products from step 52) with 0.8x VAHTS DNA clean beads, and elute in 40.5 μl Nuclease-free Water.
PAUSE POINT The RNA pre-library could be stored at 4°C for up to 72 h or at -20°C for up to 2 weeks.
58. Set up RNA library amplification reaction as follows:
5 μl RNA_droplet_N7xx (10 μM)
5 μl Illumina P5 (10 μM)
50 μl Q5 High-Fidelity 2× Master Mix
40 μl Purified DNA from above
CRITICAL STEP The RNA_droplet_N7xx primers have the RNA library sample barcodes. Use the same RNA_droplet_N7xx primer for the same sample in pre-amplification and final RNA library amplification. Use different primers for different samples if you want to sequence the samples in the same lane.
59. Performed RNA library PCR as follows:
Step Temperature Time Number of cycles
Initial denaturation 98°C 1 min 1
Denaturation 98°C 20 sec 7
Annealing 63°C 20 sec
Extension 72°C 20 sec
Final extension 72°C 1 min 1
Hold 10°C ∞ 1
60. Purify the final RNA library using 0.8× VAHTS DNA cleaning beads and elute in 20 μl Nuclease-free Water.
Library QC and sequencing Timing 4~6 d
61. Measure the concentration of each library by Qubit according to the manufacturer’s instructions.
62. Dilute the libraries to 5~10 ng/μl using Nuclease-free Water.
63. Check the library size distribution on an Agilent high-sensitivity chip according to the manufacturer’s instructions. A typical ISSAAC RNA library shows a broad peak between 250-1000 bp. A typical ISSAAC ATAC library shows a nucleosome ladder between 200-1000 bp.
64. Sequence the libraries for 150 x 8 x 16 x 150 cycles on Novaseq 6000 platform (Illumina).