GSM9737983: WT, H3K9me3 ChIP, rep3; Mus musculus; ChIP-Seq
Native ChIP-seq was performed on approximately 250,000 cells per reaction, resuspended to a concentration of ~5,000 cells/µl inl nuclear isolation buffer (Sigma, NUC-101). MNase digestion was carreid out at 21 ◦C for 8 min. Digested chromatin was then suspended in Complete IP buffer [20 mM Tris–HCl (pH 8), 2 mM EDTA, 150 mM NaCl, 0.1% Triton-X100, 1 ×Protease inhibitor cocktail, and 1 mM phenylmethanesulfonyl fluoride (PMSF)]. Following pre-clearing the chromatin on a mix of protein A / protein G beads, and after 10% was set aside for input, IP was performed overnight rotating at 30 rpm, at 4 ◦C, using 250 ng antibodies against H3K9me3 (Abcam, ab8898) and on a subset of samples also for H3K4me3 (Diagenode, C15200152). DNA was washed in low salt and then high salt washes, before eluting in ChIP elution buffer (100 mM NaHCO 3 and 1% SDS). Libraries were generated with the NEB Ultra II kit and quantitated using a Agilent Bioanalyzer. Libraries were paired-end sequenced on a NextSeq2000 using the P3 100 cycle program.