GSM5931012: sh4; Homo sapiens; ATAC-seq
50,000 intact live cells were extracted, and trypan blue was used to detect the number of live cells, 1ul of 0.4% trypan blue, 9ul of cell suspension, stained for 3-5min, and microscopic examination. Add 50ul of pre-chilled RSB (add 0.1% NP40, 0.1% Tween-20, 0.01% digitonin), invert 3 times, and incubate on ice for 3 min. Add 1ml of pre-chilled RSB (only add 0.1% Tween-20), invert 3 times and mix, centrifuge at 500xg for 5min at 4°C, discard the supernatant. Cells were lysed using cold lysis buffer (10Mm Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2 and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500 × g for 10 minutes at 4℃ and the supernatant was discarded. Immediately the pellet was resuspended in the transposase reaction mix (25 μL 2x TD buffer, 2.5 μL Transposase (Illumina) and 22.5 μL of nuclease free water). The transposition reaction was carried out for 30 minutes at 37 °C. Directly following transposition the sample was purified using a Qiagen Minelute kit. Following purification, we amplified library fragments using 1x NEBnext PCR master mix and 1.25 μM of custom Nextera PCR primers, using the following PCR conditions: 72°C for 5 minutes, 98°C for 30 seconds, followed by thermocycling at 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute，Last extension at 72℃ for 5min.The libraries were purified using a Qiagen PCR cleanup kit yielding a final library。