GSM6594225: ChIPseq.MIN6.WT.H3K27ac; Mus musculus; ChIP-Seq
Cells were trypsonized, washed with room temperature PBS twice to remove trypsin, and divided into 40 million-cell aliquots.  Cells were fixed using 1% formaldehyde (Sigma Aldrich) for 10 min at 37 °C with constant stirring to keep cells in solution, and finally quenched with 125 mM glycine for 5 min at 37 °C. Quenched cells were washed with cold PBS, and stored in 10-million aliquots at −80 °C until they were used. 10 millions cells were used per epitope in subsequent chromatin immunoprecipitations. Following nuclei extraction, chromatin was sonicated using a Covaris E220 Focused-Ultrasonicator system. Sonicated chromatin was cleared by centrifugation for 10 min at 10,000 r.p.m. (15,000 g). Soluble fractions were incubated with the indicated antibodies (see antibody list) overnight at 4°C, and captured using Protein G Dynabeads (Thermo Fisher). Antibody-chromatin complexes were captured with Protein G Dynabeads (Thermo Fisher), washed extensivley, eluted, and treated with RNAse A (Roche) for 30 min at 37 °C and Proteinase K (Life Technologies) for 3 h at 65 °C. ChIP DNA samples were extracted using SPRI beads (Beckman Coulter Agencourt AMP Xpure), washed with 80% Ethanol, and eluted using 0.1X TE. ChIP DNA samples were stored at -20C before library prep were initiated. All ChIP seq libraries were processed with Illumina's NEBNext Ultra II DNA library Prep Kit (Followed standard protocols). All ChIP seq libraries were sequenced on the Illumina Next-seq 500 with 75 bp read single-end sequencing. ChIP-sequencing libraries were prepared with Illumina's NEBNext Ultra II DNA library Prep Kit using standard protocols.