GSM5057389: RL1920_ATACseq_Dox_rep1; Homo sapiens; ATAC-seq
50,000 FACS purified cells were resuspended in 50 µl cold ATAC resuspension buffer 1 (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) NP40, 0.1% (v/v) Tween-20, 0.01% (v/v) Digitonin) and incubated on ice for 3 minutes to lyse cells. The lysis reaction was stopped by adding 1 ml of cold ATAC resuspension buffer 2 (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) Tween-20) and inverting the tube for 3 times to mix. The nuclei were pelleted by centrifugation at 500xg for 10 minutes at 4℃ in a fixed angle centrifuge. The supernatant was removed and the nuclei were resuspended in transposition mix containing 25 µl of 2x TD buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 20% (v/v) Dimethyl Formamide), 2.5 µl of 100 nM Tn5, 16.5 µl PBS, 0.5 µl of 1% (v/v) digitonin, 0.5 µl of 10% (v/v) Tween-20, and 5 µl H2O. The transposition reaction was incubated at 55°C for 30 minutes in a thermomixer with 1000 RPM mixing. Transposed DNA was cleaned up using the Qiagen MinElute PCR Purification kit following manufacturer's instructions. 20 µl of eluted DNA was subject to 8-10 cycles of PCR amplifications using NEBNext 2x MasterMix.