GSM7996338
Stable U2OS cell lines with Tet-on expression were induced with 500ng/ul Doxycycline for 4 days and processed with ChIP-seq protocol. 1x10^7 cells were cross-linked by 1% PFA for 10 min at room temperature and quenched by 125 mM glycine for 5 min. Pelleted cells were first lysed with NP40 buffer (50 mM Tris-HCl, pH 8.1, 150 mM NaCl, 5 mM EDTA, 0.5% NP40), and then resuspended with sonication buffer (20mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) for chromatin sonication by Bioruptor sonicator (Diagenode) at High output for 15 cycles (30s on, 30s off) at 4°C. Samples were centrifuged at 12000g for 10 min at 4°C and the supernatant was collected for IP. 4 μg ChIP-grade Flag antibody (Sigma, A01868-40) was added to the collected sample and incubated for 4 hours at 4°C with gentle shaking, followed by incubation of protein-G magnetic beads rocking at 4 °C overnight. Immunopreciated DNA was purified by Zymo DNA purification kit.  Library construction was prepared using the NextEra NEB library prep kit for illumina (NEB, e7370) following the manufacturer's protocol. DNA was size selected at 300-700 bp by electrophoresis and the sequencing was performed on the Illumina NOVA-seq 6000 Genome Analyzer (pair-end, 150bp). ATAC-seq libraries were prepared using the library preparing kit (Vazyme, TD501). Transposed DNA was amplified for 10 cycles, and the library DNA was further size selected with VAHTSTM DNA Clean Beads (Vazyme, N411) to the range of 150bp - 700bp./