title: GSM5397555: ATAC-seq_ESC_p13_33; Homo sapiens; ATAC-seq
library_construction_protocol: Approximately one million freshly harvested cells were pelleted and washed in PBS, then resuspended in 1 ml of RSB buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin). After 10 min incubation on ice, samples were spun at 500 g for 5 min and resuspended in 500 µl RSB without NP40 or digitonin, then strained through a 30 µm filter and pelleted again. Resulting nuclei were counted using trypan blue and 50,000 nuclei were resuspended in 25 µl of 2x TD buffer (20 mM Tris-HCl, 10 mM MgCl2, 20% dimethyl formamide) Tagmentation mix was completed by adding 100 U of loaded Tn5, 16.5 µl PBS, 0.5 µl of 1% digitonin and 0.5 µl of Tween-20 to a final volume of 50 µl, followed by incubation for 30 minutes at 37°C with 1,000 RPM mixing on a thermo block. After tagmentation, samples were cleaned up using the Qiagen MinElute PCR purification kit. Eluat was amplified using NEBNext 2x MasterMix and Nextera-based adapters as primers. After 10 PCR cycles, a double-sided bead purification was performed using 0.5x and 1.8x Ampure XP beads. Libraries were quantified by qPCR on a CFX96/C1000 cycler (Biorad) and sequenced on a Novaseq 6000 (Illumina) in 2x 61 bp paired-end format.