GSM9589230: H3K27me3 Input; Mus musculus; ChIP-Seq
H3K27me3 ChIP experiments were performed on 10T ½ cells as described previously. Briefly, the cells were cross-linked with 1% formaldehyde at room temperature for 10 minutes and the crosslinking was quenched with glycine (0.125 mol/L final concentration). Ice cold 1X PBS was used to wash the cells twice and the cells were subsequently pelleted and lysed in ChIP lysis buffer (10 mmol/L Tris-HCl, pH 8.0, 0.25% Triton X-100, 10 mmol/L EDTA, 0.1 mol/L NaCl). After lysis, the nuclei were pelleted by centrifugation and resuspended in a sonication buffer containing 1% SDS, and the chromatin was fragmented to an average size of 150-500 bp using Bioruptor Plus, from Diagenode at 10s on, 20 s off cycles. Chromatin was immunoprecipitated against H3K27me3 (Cell Signaling Technologies, #9733S) overnight, and the antibody-chromatin complexes were pulled down with Protein G Dynabeads (Invitrogen, catalogue No. 10003D). The immunoprecipitated complex was washed serially with low salt, high salt, and lithium chloride wash buffers, and crosslink reversal was performed by incubating the complex in elution buffer (1% SDS, 10 mmol/L EDTA, 50 mmol/L Tris-HCl, pH 8) overnight at 65 deg. The immunoprecipitated DNA was extracted, and library preparation was performed with the New England Biolabs Ultra II Kit according to the manufacturer's specifications.  Following size selection with AMPure XP beads to collect 200-600bp fragments, the ChIP DNA libraries were sequenced on the Illumina NextSeq platform with 75bp single-end sequencing.