GSM8290444: GM12878_H3K27ac_Low_rep2; Homo sapiens; OTHER
Sorted cells were counted and split into 50K cells per replicate, including live, unsorted control. Samples were then processed using the InTAC-seq protocol. Briefly, cells were washed once with cold PBS at 300G for 5 minutes and then once with cold PBS+0.1% Tween-20 at 300G for 5 minutes. Transposition and lysis were carried out in 1 step with each sample cell pellet being suspended with 25ul 2x TD buffer, 2.5ul Tn5 enzyme, 0.5ul 1% digitonin, 0.5ul of 10% NP40 and 21.5ul of nuclease-free water. Cell suspensions were incubated at 37C for 30 minutes on a shaker at 800RPM. After transposition, 50ul of 2X reverse crosslinking buffer (100mM N,N-Dimethylethylenediamine, pH 6.5 (Sigma Aldrich D158003), 2% SDS, 1ul Proteinase K (NEB #P8107S) in nuclease-free water) was added to cells with an overnight incubation at 37C on a shaker at 800RPM. DNA extracted from samples using QIAGEN MiniElute PCR purification columns and subsequent library preparation and next-generation sequencing were carried out as previously described in the InTAC-seq paper and this protocol is optimized from the published InTAC-seq method. For the preparation of live ATAC-seq samples from fresh cells as a comparison, samples were prepared as above, except DNA was purified immediately after transposition rather than performing crosslink reversal.