GSM5089829: HepG2 bortezomib Input [HepG2-unedited]; Homo sapiens; ChIP-Seq
Cells were dual-crosslinked with 1.5 mM ethylene glycol bis(succinimidyl succinate) (Thermo Scientific Pierce #21565) for 20 min, after which formaldehyde was added to a final concentration 1.5% and cells were incubated for another 15 min at room temperature. The crosslinking reaction was stopped by adding 0.125 M glycine. Fixed cells were treated with nuclei isolation buffer (10 mM Hepes, pH 7.9, 85 mM KCl, 0.5% NP-40, Roche Complete protease inhibitor cocktail), resuspended in ChIP lysis buffer (50 mM Tris-HCl, pH 8.0, 0.3% SDS, 10 mM EDTA, protease inhibitors) and sonicated with  Bioruptor Plus (Diagenode) for 20 cycles (30 s on, 30 s off) at 4 °C with power set to high. Sonicated chromatin was diluted 3-fold in ChIP dilution buffer (5 mM Tris-HCl, pH 8.0, 250 mM NaCl, 1.1% Triton X-100, protease inhibitors) and immunoprecipitated using mouse anti-Flag M2 monoclonal antibody (Sigma #F3165) or rabbit anti-ATF4 polyclonal antibody (Santa Cruz Biotechnology sc-200) with rotation overnight at 4 °C. Immunocomplexes were isolated by Protein G sepharose beads (Amersham Biosciences) pre-blocked in 0.05% BSA. Subsequently, chromatin crosslinking was reversed and DNA was purified using QIAquick PCR Purification Kit (Qiagen). ChIP-seq libraries were prepared from 10 ng immunoprecipitated or input DNA using the NuGEN Ovation Ultralow V2 DNA-Seq library preparation kit (NuGEN Technologies). Library DNA was size selected with SPRI beads (Beckman Coulter) to retain 200-500 bp fragments. The libraries were sequenced on an Illumina NextSeq 500 using 76 bp single-end reads.