GSM6631068: ATAC_hPGC_wk7_M_r1; Homo sapiens; ATAC-seq
Cells were sorted directly into Nuclei EZ Storage Buffer (Sigma, NUC-101) and stored at -80 °C. ATAC-seq libraries were prepared following the Omni-ATAC protocol described by Corces et al. (2017). Briefly, cells were washed in cold PBS and resuspended in cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water). Cells were then lysed in 50 μl of RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin. After lysis, 1 ml of RSB containing 0.1% Tween-20 was added. Cell pellet was resuspended in 50 μl of transposition mix (25 μl 2x TD buffer, 2.5 μl TDE1 Tagment DNA Enzyme (Nextera DNA Library Prep Kit, FC-121-1031), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water) by pipetting up and down six times. The 2X TD buffer was composed of 20 mM Tris-HCl pH 7.6, 10 mM MgCl2, 20% Dimethyl Formamide in water. Transposition reactions were incubated at 37 °C for 30 min in a thermomixer with shaking at 1,000 r.p.m. Reactions were cleaned up with MinElute Reaction Cleanup Kit (Qiagen). Tagmented DNA was amplified using the KAPA HiFi HotStart Real-Time Library Amp Kit (Roche) with modified Nextera dual indexed primers as listed in Table 1. Amplified libraries were purified using Ampure XP beads (Beckman Coulter) with double-sided size selection (1st bead selection: 0.5x; 2nd bead selection: 1.2x) according to manufacturer's protocol. Quantified and validated libraries (~ 150-1000 bp) were subjected to pair-end sequencing on HiSeq 4000 sequencing system (Illumina).