GSM2453243: C127 H3K4me3 rep1; Mus musculus; ChIP-Seq
H3K4me3 ChIP was performed by crosslinking cells in PBS with 1% formaldehyde for 15 min at 25˚C and was quenched with 150 mM glycine. For CFP1, KDM2B and RNAPII ChIP-seq, cells were fixed with 2 µM EGS for 1 h prior to the formaldehyde crosslinking. All ChIP-sequencing experiments were carried out as at least biological duplicate. Crosslinked cells were incubated in lysis buffer (50 mM HEPES KOH, pH 7.9, 140 mM NaCl, 1 mM EDTA,10% Glycerol, 0.5% NP40, 0.25% TritonX-100) for 10 minutes at 4˚C. The released nuclei were then washed (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 10 minutes at 4˚C. Chromatin was resuspended (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na Deoxycholate, 0.5% N-lauroylsarcosine), and sonicated for 50 min using a BioRuptor sonicator (Diagenode), shearing genomic DNA into 0.5–1 kb fragments. After sonication, TritonX-100 was added to a final concentration of 1.5%. Following centrifugation at 19000 g for 10 min at 4˚C, the supernatant containing soluble chromatin was isolated from the insoluble fraction. The relevant  antibodies were incubated with 750 µl of ProteinA Dynabeads (Novex) in PBS/BSA 0.5% rotating at 4˚C for 4 h. The beads were then washed three times in PBS/BSA 0.5%, to remove unbound antibody. Prior to immunoprecipitation, chromatin was diluted (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100) 10-fold (or 50-fold for H3K4me3). Immunoprecipitations were performed using 1 ml of diluted chromatin per 25 µl of antibody coated beads, and rotated overnight at 4˚C. This is equivalent to 5x10^6 cells (or 1x10^6 for H3K4me3 ChIP).  The IP was then washed one time with each of the following buffers: low salt buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.1% SDS), high salt buffer (20 mM Tris-HCl pH 8, 500 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.1% SDS), LiCl buffer (10 mM Tris-HCl pH 8, 0.25 M LiCl, 1% NP-40, 1 mM EDTA, 1% Na-deoxycholate), followed by two washes with TE buffer. Elution was performed in 100 µl of elution buffer (100 mM NaCO3, 0.1% SDS) by vigorous shaking for 30 min at 30°C. Crosslinks were reversed by the addition of 4 µl of 5M NaCl and 2 µl of 500 µg/ml RNaseA (Roche) at 65˚C followed by incubation at 42˚C for 1.5 h with 1 µl of 20 mg/ml proteinaseK. ChIP DNA was purified ChIP DNA Clean & Concentrator kit (Zymo Research). ChIP-seq libraries were generated as described previously (Blackledge et al., 2010) by the High-Throughput Sequencing centre at the Wellcome Trust Centre for Human Genetics (Oxford, United Kingdom) according to standard Illumina library generation protocol.