GSM9017384: RPMI 2650 Hypoxia ATAC-seq 2; Homo sapiens; ATAC-seq
RPMI 2650 cells (hypoxic/normoxic) were washed with PBS and harvested using TrypLE Express. For nuclei isolation, 60,000 counted cells were resuspended in 50 μL cold lysis buffer (RSB with 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin). Lysis was performed on ice for 3 min. Nuclei were then washed with 1 mL of RSB with 0.1% Tween-20, followed by centrifugation at 500 × g for 10 min at 4°C. Isolated nuclei were resuspended in 50 μL of transposition mix (Illumina TD Tagment DNA Buffer and TDE1 Tagment DNA Enzyme, with DPBS, digitonin, Tween-20, and nuclease-free water). Transposition was performed at 37°C for 30 min with shaking at 1000 RPM. Transposed DNA was cleaned up using the MinElute Reaction Cleanup Kit. Libraries were pre-amplified for 5 cycles using NEBNext® High-Fidelity 2X PCR Master Mix (NEB) with Illumina/Nextera adapter primers. Optimal additional cycles were determined by amplification profile assessment. Final libraries were purified using AMPure XP beads. Library quality was validated via Agilent High Sensitivity D1000 kit prior to high-throughput sequencing on an Illumina NovaSeq 6000 Sequencer.