GSM4118755: H9 naïve hTSC 2; Homo sapiens; ATAC-seq
Cells were harvested by TrypLE Express dissociation and centrifuged at 500 RCF for 5 minutes at 4°C. The supernatant was aspirated. Cells were washed once with cold PBS. Cell pellets were then lysed in 100 μL ATAC-seq RSB [10mM Tris pH 7.4, 10mM NaCl, 3mM MaCl2] containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin by pipetting up and down and incubating on ice for 3 minutes. 1 mL of ATAC-seq RSB containing 0.1% Tween-20 was added and mixed with the lysis reaction. Nuclei were then pelleted by centrifuging at 800 RCF for 5 minutes at 4°C. Supernatant was removed, and the nuclear pellets were resuspended in 20 μL 2x TD buffer [20mM Tris pH 7.6, 10mM MgCl2, 20% Dimethyl Formamide]. Nuclei were counted, and 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 (100nM final) (Illunina, 20034197, 16.5 μL PBS, 0.5 μL 1% Digitonin, 0.5 μL 10% Tween-20, 5 μL H2O) was then added. Transposition reactions were mixed and incubated at 37°C for 30 minutes with gentle tapping every 10 minutes. Reactions were cleaned up with the Zymo DNA Clean and Concentrator-5 kit (Zymo Research, D4014). The ATAC-seq library was prepared by amplifying for 9 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 on an Illumina NextSeq platform. QC and analysis on ATAC-seq libraries was performed using AIAP (Liu et al., 2019).
GEO Accession
GSM4118755