Sample preparation:
1. Harvest 0.2-1 million cells and digest cells into single-cell suspension.
2. Wash cells by 1 ml 0.1% BSA/PBS twice.
*Wash tubes with 1% BSA/PBS prior to use and use low-retention tubes if possible. This operation can prevent cell/beads loss.
3. Cell pellets are resuspended with 1 ml 0.1% BSA/PBS.
4. Add 7 µl 36.5% FA and incubate the tubes on ice for 5 min.
5. To stop fixation, add 14 µl 2.5M Glycine,invert the tubes several times and keep on ice for 5 min.
6. Collect at 600 g for 3 min at 4 °C and wash cell pellets with 1 ml 0.1% BSA/PBS twice.
7. The cell pellets are resuspended with 100 µl 0.1% BSA/PBS.
8. Fix cells by adding -20°C methanol drop by drop to a final concentration of 90%.
* The prepared sample can be stored at -80°C for at least six months.
Probe-1st Ab-PAT T7 complex incubation:
9. Compound stock solution in DMSO can be diluted to 10 µM in antibody buffer.
Antibody buffer:
50 mM EDTA: 40 µl;
10% BSA/PBS: 10 µl;
5% digitonin: 10 µl;
100× protease inhibitor cocktail: 10 µl;
ddH2O: adjust the volume to 1 ml.
10. 3.34 μl compound solution (10 µM), 0.5 μg anti-biotin antibody (3.33 pmol), 0.22 μl pre-assembled T7 barcoded PAT (8.25 pmol), and wash buffer (total 5 µl) are mixed thoroughly and incubated at 25℃ for 1 h.
11. Keep cells on ice for 30 min.
12. Collect cells at 1,000 g for 3 min at 4 °C.
13. Wash cells with 1 ml 0.1% BSA/PBS twice.
14. Resuspend cells with 95 µl antibody buffer with 300 mM NaCl and 5 µl probe-1st Ab-PAT T7 complex in one PCR tube, and incubate the reaction at 4°C for 4 h.
Targeted tagmentation:
15. Wash cells three times with 180 μl DIG-300 wash buffer on a tube rotator and incubate at 4°C for 5 min.
DIG-300 wash buffer:
5% digitonin: 20 µl;
1M sodium butyrate: 100 µl;
1 M HEPES pH 7.5: 200 µl;
5 M NaCl: 600 µl;
2 M spermidine: 2.5 µl;
ddH2O: adjust the volume to 10 ml.
16. Centrifuge cells at 300 g for 3 min and discard the supernatant.
17. Resuspend cells with 50 μl cold reaction buffer (10 mM MgCl2, 10 mM TAPS-NaOH pH 8.3, 0.01% digitonin, 1× protease inhibitor cocktail, 10 mM sodium butyrate).
18. Incubate the tubes at 37°C for 1 h.
*The reaction is gently mixed once after 20-min incubation.
19. Add 180 μl DIG-300 wash buffer (0.01% Triton X-100 and 5 mM EDTA) to each tube, mix well, and rotate for 10 min.
20. Wash cells three times with 180 μl DIG-300 wash buffer.
21. Centrifuge cells at 300 g for 3 min and discard the supernatant.
Second 1st Ab-PAT T7 incubation and tagmentation:
22. 0.5 μg antibody (e.g. antibodies against histone modifications or chromatin binding proteins) (3.33 pmol), 0.22 μl pre-assembled T7 barcoded PAT (8.25 pmol), and wash buffer (total 5 µl) are mixed thoroughly and incubated at 25℃ for 1 h. Resuspend cells with 95 µl antibody buffer with 300 mM NaCl and 5 µl 1st Ab-PAT T7 complex in one PCR tube, and incubate the system at 25°C for 1 h.
23. Repeat steps 15-21 to achieve the second tagmentation.
Binding 2nd antibody:
24. Resuspend cells with 100 μl cold antibody buffer premixed with 2nd antibody in a 1:500 dilution.
25. Place the tubes on a rotator at 4°C for 30 min.
26. Wash cells twice with 180 μl DIG wash buffer and discard the supernatant.
Binding PAT-T5 and targeted tagmentation:
27. Add 100 µl DIG-300 wash buffer (0.01% Triton X-100, 1× protease inhibitor cocktail, and 10 mM sodium butyrate) containing sample-barcoded 9 µg/ml pre-assembled T5 PAT.
* Different samples are differentiated with different pre-assembled T5 barcoded PAT.
28. Place the tubes on a tube rotator and incubate at 4°C for 1 h.
29. Centrifuge at 300 g for 3 min.
30. Wash cells twice by 180 µl DIG-300 wash buffer (0.01% Triton X-100) on a tube rotator and incubate at 4°C for 5 min.
31. Resuspend cells with 50 μl cold reaction buffer.
32. Incubate the plate at 37°C for 1 h.
*The reaction is gently mixed once after 20-min incubation.
33. Add 180 μl DIG-300 wash buffer (0.01% Triton X-100 and 5 mM EDTA) to each PCR tube, mix well, and rotate for 10 min.
34. Wash cells three times with 180 μl DIG-300 wash buffer (0.01% Triton X-100).
35. Centrifuge the cells at 300 g for 3 min and discard the supernatant.
(Optional) Chromatin accessibility:
36. Cells are suspended in 50 μl of tagmentation mix, which consists of 33 mM Tris-acetate (pH 7.8), 66 mM potassium acetate, 10 mM magnesium acetate, 16% dimethylformamide, 0.01% digitonin, and 2.5 μM of Tn5-S5/S7 transposome complex.
37. The mixture is incubated at 30 °C for 30 min.
38. To stop the reaction, an equal volume of tagmentation stop buffer (containing 10 mM Tris-HCl pH 7.8, 20 mM EDTA pH 8.0, and 2% BSA) is added.
Two round hybridizations:
39. 150 µl NSB buffer is added to each PCR tube and cells are centrifuged at 300 g for 3 min to remove the supernatant.
NSB buffer:
1 M Tris-HCl pH 7.5: 500 µl;
5 M NaCl: 100 µl;
5% TX-100: 100 µl;
1 M MgCl2: 150 µl;
10% BSA/PBS: 500 µl;
ddH2O: adjust the volume to 50 ml.
40. Cells are washed with 180 µl NSB buffer for three times.
41. Cells are mixed and resuspended in 5 ml of hybridization mix (1 × T4 ligase buffer, 0.01 % Triton X-100, and 0.25 × NSB buffer).
42. Cells in hybridization mix (40 μl) are added to each of the 96 wells in the 1st round barcoding plate, in which each well already contain 10 μl round 1 adaptor.
43. The round 1 barcoding plate is incubated for 30 min at room temperature with gentle shaking (300 rpm) to allow hybridization to occur before adding blocking oligos.
44. 10 μl of round 1 blocking oligos is added followed by incubation for 30 min at 25℃ with gentle shaking (300 rpm).
45. Cells from all 96 wells are combined and redistributed.
46. Cells in hybridization mix (50 μl) are added to each of the 96 wells in the 2nd round barcoding plate, in which each well already contains 10 μl round 2 adaptors (total 60 μl).
47. The round 2 barcoding plate is incubated for 30 min at room temperature with gentle shaking (300 rpm) to allow hybridization to occur before adding blocking oligos.
48. 10 μl of round 2 blocking oligos is added followed by incubation for 30 min at 25℃ with gentle shaking (300 rpm).
49. After adding the round 2 blocking oligos, cells from all wells are combined and centrifuged at 1,000 g for 3 min to remove the supernatant.
Ligation:
50. Cells are washed twice with 1 ml NSB buffer, and centrifuged at 600 g for 3 min.
51. Cells are resuspended in the 200 µl ligation mix (1x T4 ligation buffer, 20U/μl T4 DNA ligase (M0202L, NEB), and 0.05% Triton X-100 and 0.2 × NIB buffer) and incubated for 30 min at 25℃ with gentle shaking (300 rpm).
Redistributing cells and Releasing DNA:
52. Wash cells twice with cell suspension buffer (3 mM MgCl2, 0.01% Triton X-100 and 0.1% BSA/PBS).
53. Cells are filtered through cell strainer to remove cell clumps.
54. Count the cell number and resuspend the cells at the concentration of 1,000-5,000 cells/µl by 0.1% BSA PBS.
55. 1 µl of cells are sorted into each well of a new 96-well plates, containing 4 µl lysis buffer (10 mM Tris-HCl pH 8.5, 0.05 % SDS and 0.1 mg/ml proteinase K) in each well).
56. Incubate the plate at 55°C for 15 min.
57. Add 1 µl 10 mM PMSF and 1 µl 1.8% Triton X-100 to each well and incubate the plate at 37 °C for 10 min to quench SDS.
Amplification of transposed fragment:
58. A total of 50 μl of PCR mix is added to each well, comprising 10 μl 5× KAPA HiFi buffer, 1 μl 10 mM dNTP Mix, 2 μl 10 µM P7 connector primer, 2 μl 10 µM TruSeq P5 primer, 0.5 μl 1 U/μl KAPA HiFi HotStart DNA polymerase, 1 μl 25 mM MgCl2, and 26.5 μl ddH2O.
59. PCR enrichment is conducted with one cycle of 72 °C for 5 min, one cycle of 95 °C for 3 min, and twelve cycles of 98 °C for 20 s, 65 °C for 30 s, and 72 °C for 30 s, followed by one cycle of 72 °C for 5 min and a hold at 4°C.
60. 45 μl (0.9×) of custom AMPure XP beads are added to each well and mixed thoroughly, and DNA is purified and eluted in 10 μl of ddH2O.
61. For double size selection purification, 0.5× + 0.4× of AMPure beads are used.
62. DNA is purified and eluted with 25 μl ddH2O, followed by addition of 25 μl of PCR mix containing 10 μl 5× KAPA HiFi, 1 μl 10 mM dNTP Mix, 2.5 μl 10 µM P7 primer, 2.5 μl 10 µM P5 primer, and 0.5 μl 1 U/μl KAPA HiFi HotStart DNA polymerase, 1 μl 25 mM MgCl2 and ddH2O.
63. The second PCR enrichment is carried out with one cycle at 72 °C for 5 min, one cycle at 98 °C for 3 min, six cycles at 98 °C for 20 s, 65 °C for 30 s, 72 °C for 1 min, and one cycle at 72 °C for 1 min, followed by hold at 4 °C.
64. Finally, the library is purified once with 0.9× AMPure XP beads, and the DNA part library is obtained after purification and elution with ddH2O.