GSM8862431: A172 glioblastoma cell line ATAC-seq rep2; Homo sapiens; ATAC-seq
Cells were treated with Trypsin-EDTA 0.05% and washed with DPBS (ThermoFisher 14190-169). Cell viability and concentration were assessed by the LUNA-FL Dual Fluorescence Cell Counter. Per cell line, 80,000 cells were used to perform ATAC-seq. After centrifugation at 500 g for 5 min at 4°C, the supernatant was replaced with 50 µl ice cold ATAC-seq lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630, 0.1% Tween-20, 1% BSA and 0.01% Digitonin) and the cells were mixed by pipetting. After incubation for 5 min on ice, 1 ml of wash buffer (20 mM Tris-HCl, 20 mM NaCl, 6 mM MgCl2, 0.1% Tween-20, and 1% BSA) was added to the lysed cells and mixed gently by inverting 3 times. After centrifugation at 500 g for 5 min at 4°C, supernatant was removed and the nuclei were resuspended in 50 µl ATAC mix (10 mM Tris-HCl, 10% dimethylformamide, 5 mM MgCl2, 1x DPBS, 0.1% Tween-20, 0.01% Digitonin and 3.75 ng/µl Tn5 enzyme) and incubated for 1 hour at 37°C. Transposed DNA was purified using a QIAGEN MinElute purification column and eluted in 15 µl elution buffer (Qiagen). The ATAC-seq library was completed by amplifying the transposed DNA in a total volume of 50 µl PCR mix (15 µl purified DNA, 25 µl NEBNext High Fidelity PCR Master Mix (NEB), 2.5 µl FWD and 2.5 µl REV primer) with the following program: 72°C for 5 min, 98°C for 30 s, followed by 10 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min). The final libraries were subjected to 0.4x-1.2x doublesided Ampure purification and eluted in 20 µl elution buffer (Qiagen).