GSM4647511: Mouse Embryonic Fibroblasts - BRD4-T20; Mus musculus; ChIP-Seq
MEFs (~ 5 x106 in 15-cm plate) were fixed with 1% formaldehyde (Sigma) in buffer A (50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.5 mMEGTA) for 10 min and quenched with 0.125 M glycine for 5 min. Cells were washed and incubated in nucleus isolation buffer (50 mM HEPES, pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25 % Triton x-100). Nuclear pellets (~2 x 107) were re-suspended in 1 ml shearing buffer (1 x TE, pH 8, and 0.1% SDS) and transferred to Covaris millitubes and sonicated in Covaris ME200 sonicator. Sonicated chromatin was precipitated with anti-BRD4 antibody (IgG) pre-bound to Dynabeads Protein G (Thermo Fisher). Immunoprecipitated DNA was de-crosslinked, digested with proteinase K, and purified using QIAquick PCR Purification Kit (Qiagen). Precipitated DNA was validated by qChIP. Precipitated DNA (~ 20 ng) was used for library construction using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolab). Library DNA with fragment length of 200-400 bp was subjected to single end sequencing on a Nextseq system (Illumina).