GSM7656797: HepG2_Ctrl_1, ATAC-seq; Homo sapiens; ATAC-seq
Tazemetostat (EPZ-6438)-treated 50,000 of HepG2 cells were pretreated with 200 U/ml DNase (Worthington) for 30 minutes at 37 °C to remove free-floating DNA and digest DNA from dead cells. The cells were then washed, resuspended in cold phosphate-buffered saline (PBS). Cells were harvested from culture plate using TrypLE (12604013; Gibco) and resuspended in 1 mL of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.5 (15567-027; Invitrogen), 10 mM NaCl (AM9759; Invitrogen), and 3 mM MgCl2 (AM9530G; Invitrogen) in nuclease-free water), Cells were centrifuged at 500 relative centrifugal force (RCF) for 5 minutes in a pre-chilled (4 °C) fixed-angle centrifuge. After centrifugation, the supernatant was carefully aspirated to avoid the cell pellet, and the cells were then resuspended in 50 μl of RSB containing 0.1% NP40 (11332473001; Roche), 0.1% Tween-20 (11332465001; Roche), and 0.01% digitonin (G9441; Promega). This cell lysis reaction was incubated on ice for 3 minutes. After lysis, 1 ml of RSB containing 0.1% Tween-20 (without NP40 or Digitonin) was added, and the tubes were inverted to mix. Nuclei were then centrifuged for 10 minutes at 500 RCF in a pre-chilled (4 °C) fixed-angle centrifuge. The supernatant was removed, and nuclei were resuspended in 50 μl of transposition mix (25 μl 2× TD buffer, 2.5 μl transposase26 (100 nM final), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water). The transposition reactions were incubated at 37 °C for 30 minutes in a thermomixer with shaking at 1,000 revolutions per minute (RPM). Reactions were cleaned up with MinElute Reaction Cleanup Kit (28206; Qiagen). Reactions were pre-amplified for 5 cycles using NEBNext High-Fidelity 2x PCR MasterMix (M0541L; NEB) with adapter primers. The amplification profiles were manually assessed to determine the number of additional cycles to amplify as previously described 52. The final amplified library samples were stored at -80 °C for ATAC-seq after library purification with AMPure XP (A63880; Beckman). Libraries were validated using the Agilent High Sensitivity D1000 kit (5067-5584, 5067-5585; Agilent) and were subjected to high-throughput sequencing using the Illumina HiSeq 2500 Sequencer.