GSM5833884: E7_DMSO_d4_H3Ac; Homo sapiens; ChIP-Seq
About 5-30x10^6 HUDEP-2 cells were cross-linked with fresh 1% formaldehyde (Fisher Scientific) with/without dTAG-47 for 6 min at room temperature (RT) and quenched with 1.25 M glycine for 5 min at RT. Cells were then washed three times in cold phosphate-buffered saline (PBS) containing protease inhibitors and cell pellets flash-frozen in LN2. Nuclei were isolated using the truChIP kit (Covaris Inc.), and chromatin was sheared at 4°C, 140 W, and 200 cycles per burst with 10% duty factor for 8 min (Covaris). Input fraction was saved (1%), and the remaining sheared chromatin was used for ChIP with antibodies specific for an epitope or an immunoglobulin G isotype control (Millipore) in immunoprecipitation buffer [0.1% Triton X-100, 0.1 M tris-HCl (pH 8), 0.5 mM EDTA, and 0.15 M NaCl in 1× Covaris D3 buffer] at 4°C, rotating overnight, followed by incubation with Dynabeads Protein G (10003D, Life Technologies) for 4 hours. The chromatin-bead-antibody complexes were then washed sequentially with three wash buffers of increasing salt concentrations and TE buffer. Chromatin was then eluted using 1% SDS in Tris-EDTA. Cross-linking was reversed by incubation with ribonuclease A (Roche) for 1 hour at 37°C, and with Proteinase K (Roche) at 65oC overnight. DNA was purified with the QIAquick PCR Purification Kit (28104, Qiagen). ChIPseq libraries were prepared using Swift S2 Acel reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1ng of DNA according to manufacturer's protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by Qubit fluorometer and Agilent TapeStation 2200.  Library pooling and indexing was evaluated with shallow sequencing on an Illumina MiSeq. Subsequently, libraries were sequenced on an Illumina NovaSeq6000 targeting 40 million 150bp read pairs by the Molecular Biology Core facilities at Dana-Farber Cancer Institute.