GSM9885957: SAS cells, siControl, ATAC-seq, biological replicate 1 (A2-19); Homo sapiens; ATAC-seq
Cells were centrifuged at 300 × g for 5 min and the supernatant was removed. Twice the pellet volume of Corning Cell Recovery Solution (Corning, 354253) was added and samples were incubated on ice for 1 h. Cells were centrifuged at 300 × g for 5 min, washed with ice-cold 1× PBS, and gently lysed in 45 µL ice-cold lysis buffer containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% digitonin, 0.1% Tween-20, and 0.3% BSA. Nuclei were pelleted at 500 × g for 10 min at 4°C, washed twice with 45 µL wash buffer containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, and 0.3% BSA, and inspected microscopically. Nuclei were resuspended in 5 µL transposition mix containing 2.5 µL 2× Tagment DNA Buffer, 0.5 µL Tagment DNA Enzyme (Illumina, 20034210), and 2 µL wash buffer, incubated at 37°C for 60 min, and DNA was purified using the MinElute Reaction Cleanup Kit (QIAGEN, 28206). Ten microliters of tagmented DNA were pre-amplified in a 24 µL PCR containing 1× KAPA HiFi HotStart ReadyMix (Roche, 7958935001) and 0.08 µM unique dual Nextera indexing primers. Cycling conditions were 72°C for 5 min, 98°C for 30 s, and 5 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min. One microliter of pre-amplified product was used in qPCR to determine the final amplification cycle number; eight additional cycles were applied. Libraries underwent XP bead size selection (Beckman Coulter, A63882) using first 1.55× and then 0.6× bead-to-sample ratios, were quantified using the Fragment Analyzer (Agilent), and were sequenced on an Illumina NovaSeq 6000 in 2 × 100 bp paired-end mode at an average depth of approximately 50 million read pairs per library.