TITLE: Ten(10)X-compatible Combinatorial Indexing ATAC sequencing (txci-ATAC-seq)
AUTHORS: Hao Zhang, Ryan Mulqueen, Andrew Adey, Darren Cusanovich

[DESCRIPTION]
The txci-ATAC-seq method is a large-scale single-cell ATAC-seq technique that combines the Tn5-based pre-indexing with the 10X Chromium-based microfluidic barcoding. This molecular hashing strategy enables the profiling of up to 200,000 nuclei across multiple samples in a single emulsion reaction.

[STEPS]
SECTION: txci-ATAC-seq: Preparing nuclei
20. Add 3 ml RSB washing buffer to an empty 15 ml tube for each sample.

SECTION: txci-ATAC-seq: Preparing nuclei
21. Transfer 1 ml nuclei stored in the freezing buffer to the 15 ml tube containing 3 ml RSB washing buffer.

SECTION: txci-ATAC-seq: Preparing nuclei
22. Pellet the nuclei at 500 RCF for 10 min at 4°C.

SECTION: txci-ATAC-seq: Preparing nuclei
23. Resuspend nuclei with 1 ml RSB washing buffer and then transfer to a 1.5 ml LoBind tube through Flowmi (40 micron).
24. Pellet the nuclei at 500 RCF for 5 min at 4°C in a fixed-angle centrifuge.
SECTION: txci-ATAC-seq: Preparing nuclei
19. Take out flash-frozen nuclei (~3 million in 1 ml) from liquid nitrogen for each sample and thaw in a water bath at 37°C for about 1 min.
SECTION: Loading Tn5
1. Resuspend Tn5ME-A, Tn5ME-B, and Tn5MErev in the annealing buffer to a final concentration of 100 μM.
SECTION: Loading Tn5
2. Prepare annealed linker A: Mix one volume of Tn5ME-A with one volume of Tn5MErev in a PCR tube. 
e.g. 100 μl Tn5ME-A + 100 μl Tn5Merev.
SECTION: Loading Tn5
3. Prepare annealed linker B: Mix one volume of each barcoded Tn5ME-B with one volume of Tn5MErev on a 96-well plate.
e.g. 10 μl Tn5ME-B (Index A1) + 10 μl Tn5MErev.
  
4. Mix briefly by pipetting and run the following PCR program in a thermocycler for annealing oligos.
 

5. Add 1 μl of each annealed linker (A and B) to 20 μl of the Tn5 stock (0.3 mg/ml) on a 96-well plate with a unique annealed linker B in each well.
25. Resuspend nuclei with 100 μl of 1X PBSB for each sample.
26. Count nuclei with DAPI:
26.1. Add 1 μl of 300 μM DAPI to 100 ul nuclei;
26.2. Incubate on ice for 5 mins;
26.3. Add 10 μl stained nuclei to the countess slide to count nuclei.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
28. Thaw a 96-well plate preloaded with 5 μl of barcoded Tn5 on ice. Mix by brief shaking at 1400 rpm for 30 seconds, spin for a minute at 2000 RCF at 4°C, and carefully unseal the aluminum foil seal.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
27. Prepare TD mix:

 

 .

SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
29. Dilute nuclei to 2857 nuclei/μl in PBSB and then mix 7 μl diluted nuclei with 13 μl TD mix for each well.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
30. Add 20 μl nuclei/TD mix mixture to each well of the 96-well plate containing 5 μl of barcoded Tn5 per well (total 25 μl).
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
31. Seal the plate using Bio-Rad Microseal B film.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
32. Mix by shaking at 1000 rpm for one minute.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
33. If liquid splashes to the seal, briefly spin at 500 RCF for 10 sec.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
34. Incubate at 37°C for 60 min in a thermocycler block with a heated lid (47°C).
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
35. Thaw TMG washing buffer on ice.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
36. Remove the plate from the thermocycler.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
37. Briefly centrifuge at 500 RCF for 10 sec at 4°C.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
38. Incubate the plate on ice for 5 min.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
39. Pool nuclei in a LoBind 12-tube strip and then transfer them to a 15 ml conical tube preloaded with 400 ul of TMG.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
40. Add 50 μl/well of TMG to the first row of the plate and pipette them throughout the whole plate to wash out the residual nuclei remaining in the plate.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
41. After washing the last row of the plate, the TMG was transferred to the same conical tube that was used to collect the barcoded nuclei.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
42. Centrifuge nuclei at 500 RCF for 10 min at 4°C.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
43. Remove most of the supernatant.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
44. Resuspend nuclei with 500 μl of TMG, then transfer to a 1.5 ml LoBind Tube through Flowmi.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
45. Centrifuge at 500 RCF for 5 min at 4°C.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
46. Remove most of the supernatant and resuspend the nuclear pellet with 30 μl of LBS.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
47. Count nuclei with a hemocytometer.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
48. Take the volume of solution containing the desired number of nuclei and dilute it with the LBS to make a total of 15 μl.
SECTION: txci-ATAC-seq: 96 barcoded Tn5 transposition
49. Use the 15 μl dilution as input into the 10X Chromium droplet generator – follow Step 2, page 24 of the Chromium Single Cell ATAC kit instructions (10x Document CG000209 Rev D) to complete the assay.
SECTION: txci-ATAC-seq: Modification of 10X Chromium protocol
50. For Step 2.5. GEM Incubation:
SECTION: txci-ATAC-seq: Modification of 10X Chromium protocol
50.1. a. Incubate in a thermal cycler with the following protocol (Lid temperature at 105℃).

 

SECTION: txci-ATAC-seq: Modification of 10X Chromium protocol
50.2. b. Store at 15°C for up to 18 h or at −20°C for up to 1 week, or proceed to the next step.
SECTION: txci-ATAC-seq: Modification of 10X Chromium protocol
51. For Step 4.1 Sample Index PCR
SECTION: txci-ATAC-seq: Modification of 10X Chromium protocol
51.1. c. Add 2.5 μl of customized i7 TruSeq primer (25 μM) containing an 8 bp custom barcode to each 10X library. Record assignment. Pipette mix and centrifuge briefly.
  
SECTION: txci-ATAC-seq: Modification of 10X Chromium protocol
51.2. d. Incubate in a thermal cycler with the following protocol (Lid temperature at 105℃).

 

SECTION: Sequencing
52. 
 

6. Mix briefly by pipetting, and then incubate at 23°C for 30 minutes in a thermomixer at 350 rpm.
7. Store at -20°C.
SECTION: Isolation of nuclei from cell lines
8. Remove approximately 10x106 cells from culture.

 

 

SECTION: Isolation of nuclei from cell lines
9. Pellet the cells at 500 RCF at 4°C for 5 min in a swinging-bucket centrifuge.
SECTION: Isolation of nuclei from cell lines
10. Aspirate supernatant.
SECTION: Isolation of nuclei from cell lines
11. Resuspend pellet in 200 µl RSB Lysis Buffer.
SECTION: Isolation of nuclei from cell lines
12. Incubate on ice for 3 minutes.
SECTION: Isolation of nuclei from cell lines
13. Add 1 ml RSB Washing Buffer.
SECTION: Isolation of nuclei from cell lines
14. Take 10 µl nuclei and dilute it with 40 µl of Omni TD buffer, then count the nuclei on a hemocytometer by adding 50 µl Trypan blue solution to the diluted nuclei (Note: we found that adding the RSB-resuspended nuclei straight to Trypan blue solution will cause inflation of nuclei, and diluting nuclei in Omni TD buffer before exposure to Trypan blue improves the nuclei integrity).
SECTION: Isolation of nuclei from cell lines
15. Pellet the remaining nuclei in RSB Washing Buffer at 500 RCF for 10 min at 4°C in a fixed-angle centrifuge.
SECTION: Isolation of nuclei from cell lines
16. Resuspend nuclei pellet in FBW at ~3 million nuclei/ml.
SECTION: Isolation of nuclei from cell lines
17. Snap-freeze nuclei in liquid nitrogen, and then transfer the cryovials to a liquid nitrogen dewar (or -80°C) for long-term storage.
SECTION: Isolation of nuclei from lung tissue
18. The following protocol can be used to isolate nuclei from lung tissues.