GSM9685277: WT_HDP2_day8; Homo sapiens; ATAC-seq
ATAC-seq was performed following a published protocol (Buenrostro, 2013) using the Illumina Tagment DNA Enzyme and Buffer Kit. 10,000-50,000 cells were used per biological replicate. Cells were lysed and nuclei were isolated prior to tagment reaction for 30 minutes at 37℃. Transposed DNA fragments were purified using a MinElute Kit (Qiagen). ChIPmentation experiments were performed as previously described (Schmidl, 2015) with modifications. On day 8 of erythroid differentiation, 100,000-1,000,000 HUDEP-2 cells were fixed with 2 mM disuccinimidyl glutarate (DSG) for 30 minutes, followed by 1% formaldehyde for 10 minutes, then quenched with 125 mM glycine. Fixed cell pellets were washed with PBS, then lysed in cold lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA and 0.5% SDS) for 30 minutes on ice. The lysate was sonicated with the Covaris ME220 Focused-ultrasonicator to shear the DNA into ~200-300 bp fragments. The sonicate was neutralised with 1% Triton X-100 and incubated overnight with Dynabeads protein A and G (Invitrogen) and 5 μg GATA1 antibody (Abcam, ab181544). Chromatin-bound beads were washed three times using a low salt, a high salt and a LiCl-containing wash buffer, followed by on-bead tagmentation of the immunoprecipitated chromatin using Tagment DNA enzyme (Illumina). Samples were washed with 10 mM Tris-HCl before library preparation. Libraries were amplified and indexed using the Illumina Nextera index primers by PCR. Profiles were assessed using D1000 ScreenTape Assays (Agilent) and quantified using NEBNext Library Quant Kit for Illumina (NEB).