GSM8181079: shADA2B-5d-Dox-1; Homo sapiens; ATAC-seq
The Tn5 transposition reaction was performed using the Illumina Tagment DNA TDE1 Enzyme and Buffer Kit (Illumina). For each sample 100,000 cells were collected and washed with cold PBS. A resuspension buffer (10 nM Tris-HCl, pH7.5, 10 mM NaCl, 3 mM MgCl2) was used for subsequent steps. Cells were lysed in 100 μl lysis buffer (0.1% NP-40, 0.1% Tween-20, and 0.01% Digitonin in resuspension buffer) on ice for 3 minutes, then washed with 1 ml wash buffer (0.1% Tween-20 in resuspension buffer). Lysates were cleared by centrifugation at 500g, 4°C for 10 minutes. The nuclear pellet was resuspended in 100 μl transposition reaction mix (50 μl Tagment DNA Buffer, 33 μl PBS, 1 μl 10% Tween-20, 1 μl 1% Digitonin, 5 μl TDE1, 10 μl nuclease-free water) and incubated at 37°C on a thermomixer with 1,000 rpm for 30 minutes. DNA was isolated using the Qiagen MinElute Reaction Cleanup Kit (Qiagen). Library was prepared with NEBNext High-Fidelity 2X PCR Master Mix (NEB) and SYBR Green I (ThermoFisher).