GSM7235224: ARPE19 cells, ATAC-Seq, normal control; Homo sapiens; ATAC-seq
Here we used Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling. 50,000 cells were lysed in 1 ml of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2) for 3 min, and then centrifuged at 500 r.c.f. for 5 min at 4°C. Cell pellets were resuspended in 50 μl of ATAC-seq RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin, and then incubated on ice for 3 min. After lysis, 1 ml of ATAC-seq RSB containing 0.1% Tween-20 (without NP-40 or digitonin) was added and mixed well. Nuclei were then centrifuged at 500 r.c.f. for 10 min at 4 °C. Nuclei were resuspended in 50 μl of transposition mix containing 25 μl of 2× TD buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl2 and 20% Dimethyl Formamide), 2.5 μl of transposase (100 nM final), 16.5 μl of PBS, 0.5 μl of 1% digitonin, 0.5 μl of 10% Tween-20, and 5 μl of sterile H2O. Transposition reactions were incubated at 37 °C for 30 min in a thermomixer with shaking at 1,000 r.p.m. Transposed DNA was purified with MinElute PCR Purification Kit (Qiagen) and eluted in 10 μl of EB buffer. End repair, A adding, adaptor adding reaction and PCR amplification were performed sequentially. The libraries were sequenced by the Illumina PE150 paired-end sequencing platform.