1. Spin down at 400 g for 5 min. Remove supernatant.
2. Resuspend in 1000uL Wash Buffer.
3. Spin down cells at 400 g for 5 min. Remove supernatant.
4. Re-suspend in 200-1000 uL Wash buffer depending on size of the cell pellet with RNase Inhibitor. Count cells at this point – take 10uL of trypan blue and 10uL of sample and determine starting concentration of sample. If there is more than 1500 cells/uL then add more wash buffer and recount.
5. Aliquot cell suspension to load approximately 10,000 cells/ul (refer to 10X 3’ NEXT GEM protocol, Figure 2). Set aside another 50-100 uL of cell suspension for back up. Centrifuge remaining sample at 400g for 8’ at 4°C.
6. While remaining cell suspension is being spun down, load 10X CHIP G (single-cell). After loading chip you have 17 minutes to process remaining sample for sn-ATAC.
7. After spin is finished, re-suspend cells in 500 L of cold lysis buffer. Pipette gently for 25 times (until pellet is fully resuspended). Incubate on ice for 2 min.
8. Add 400 uL Wash Buffer to same tube.
9. Transfer the clear lysis-wash buffer suspension using the 40um flowmi cell strainer into a 2.0 mL tube. Centrifuge sample at 400g for 5’ at 4°C.
10. Once the sample is finished spinning, remove as much supernatant as possible. The pellet should be at the very bottom of tube if using ST8 Centrifuge. Add 7-10uL 1X Nucleus Dilution Buffer (sn-ATAC-Seq submission buffer). Gently resuspend until nuclei are completely re-suspended. Count nuclei at this point – Add 8 uL 1X Nucleus Dilution Buffer to 2 uL of sample and 10 uL of trypan blue. If concentration of nuclei is greater than 3,000 nuclei/uL then proceed with ATAC prep.
11. Continue both 10X protocols accordingly.
a. Chromium Single Cell ATAC Reagent Kits User Guide (v1 Chemistry)
b. Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry)