GSM6081189: ATAC_seq_PIS_5; Homo sapiens; ATAC-seq
Frozen tissue was digested and grinded by trypsin and washed three times in PBS. Then the broken tissue was centrifuged and resuspended in lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) for 10 minutes. Next, the cells after lysis were centrifuged at 500 g at 4°C for 5 minutes. Carefully discarded the supernatant and the sediment were nuclei. Nuclei were counted after addition of trypan blue, which stains all nuclei due to membrane permeabilization from freezing. 50,000 counted nuclei were then transferred to a tube containing 1 ml of ATAC-seq RSB with 0.1% Tween-20. Nuclei were pelleted by centrifugation at 500r.c.f. for 10 min in a pre-chilled (4°C) fixed-angle centrifuge. Supernatant was removed using the two pipetting steps described above. Because the nuclei were already permeabilized, no lysis step was performed, and the transposition mix (25μl 2× TD buffer, 2.5μl transposase (100nM final), 16.5μl PBS, 0.5μl 1% digitonin, 0.5μl 10% Tween-20, 5μl water) was added directly to the nuclear pellet and mixed by pipetting up and down six times. Transposition reactions were incubated at 37°C for 30 min in a thermomixer with shaking at 1,000 r.p.m. Polymerase chain reaction amplification and size selection (150-500 bp) were performed using Agencourt AMPure XP (Beckman Coulter) and Bioanalyzer 2100 (Agilent). The library concentration and quality were assayed using the 2100/2200 Bioanalyzer (Agilent) prior to sequencing. Libraries were pooled at equimolar ratios with barcodes and sequenced on the BGISEQ-500 platform (BGI, Shenzhen, China).