GSM5121154: Re-Priming_BRG1_KO_rep2; Homo sapiens; ATAC-seq
Cells were harvested by TrypLE Express dissociation and centrifuged at 500 RCF for 5 min at 4 ℃. After aspirating supernatant, cells were washed once with cold PBS. Cell pellets were resuspended in 100 ul ATAC-seq RSB [10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2 in water] consisting of 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin by pipetting up and down and incubating on ice for 3 min. After lysis, 1 mL of ATAC-seq RSB containing 0.1% Tween-20 was added and inverted with the lysis reaction. Then, nuclei were pelleted by centrifugation at 800 RCF for 5 min at 4 ℃. Supernatant was removed, and the nuclei were resuspended in 20 µL 2x TD buffer [20 mM Tris pH 7.6, 10 mM MgCl2, 20% Dimethyl Formamide]. 50,000 counted nuclei were then transferred to a tube with 2x TD buffer filled up to 25 µL. 25 µL of transposition mix [2.5 µL Transposase (100 nM final) (Illunina, 20034197, 16.5 µL PBS, 0.5 µL 1% digitonin, 0.5 µL 10% Tween-20, and 5 µL water) was added. Transposition reactions were mixed and incubated for 30 min at 37℃ with gentle tapping every 10 min. Reactions were cleaned up with the Zymo DNA Clean and Concentrator-5 kit (Zymo Research, D4014). The ATAC-seq library was amplified for nine cycles on a PCR machine. The PCR reaction was purified with Ampure XP beads (Beckman Coulter, A63880) using double size selection following the manufacturer's protocol, in which 27.5 µL beads (0.55X sample volume) and 50 µL beads (1.55X sample volume) were used based on 50 µL PCR reaction. The ATAC-seq libraries were quantitated by Qubit assays and sequenced by an Illumina NextSeq platform. QC and analysis on ATAC-seq libraries was performed using AIAP (Liu et al., 2019).