GSM8240300: TCRseq_WT_12J8h_rep1; Homo sapiens; OTHER
Cells were crosslinked with 0.5 mg/mL disuccinimidyl glutarate (DSG; Thermo Fisher) in PBS for 45 min at room temperature. Cells were washed once with PBS followed by incubation with 1% PFA for 20 min at RT. Fixation was stopped by addition of glycine in PBS to a final concentration of 0.1 M for 3 min at RT. This was followed by washing with cold PBS and collection of the cells in 0.25% Triton X-100, 10 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), and 20 mM Hepes (pH 7.6) in miliQ. Chromatin was pelleted by centrifugation for 5 min at 400 × g and incubated in 150 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 50 mM Hepes (pH 7.6) in miliQ for 10 min at 4 °C. Chromatin was again pelleted by centrifugation and resuspended in ChIP-buffer (0.15 % SDS, 1 % Triton X-100, 150 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 20 mM Hepes (pH 7.6) in miliQ) to a final concentration of 15 × 106 cells/mL. Chromatin was sonicated to approximately one nucleosome using the Bioruptor Pico (Diagenode), 8 cycles of 30 s ON/30 s OFF in a 4°C water bath. ChIP was performed on 280-300ul sonicated material (~25 µg of chromatin per sample) with 3 µg of rat anti-RNAPII-S2 antibody (Millipore, clone 3E10) by overnight incubation at 4 °C, followed by a 1.5 h protein-chromatin pull-down with 20 µl protein G Dynabeads (Thermo Fisher, 10003D). ChIP samples were washed extensively, followed by decrosslinking for 4 h at 65 °C in the presence of proteinase K. DNA was purified using a Qiagen MinElute kit. Sample libraries were prepared using Hifi Kapa sample prep kit and A-T mediated ligation of Nextflex adapters or xGen UDI-UMI adapters. Samples were sequenced using an Illumina NextSeq500 or HiSeq X, using paired-end sequencing with 42 or 151 bp from each end.