GSM1384944: CDP_H3K9me3; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed as described before (Dahl and Collas, 2008) with minor modifications. Briefly, sorted cells were cross-linked at a concentration of 2 million cells/ml with 1% formaldehyde for 6 min at room temperature. Cross-linking was stopped with 0.125 M glycine. Chromatin sonification into fragments of 200-400 bp in size was done by Diagenode Bioruptor with cooling device at 4°C for 10 min with 30 s pulse/pause cycles. Sheared lysates were clarified by centrifugation at 12,000g (10 mins, 4°C). 10 µl Dynabeads Protein A (Life Technologies) were preincubated with either 1 µg IgG control (Santa Cruz) or specific antibodies for H3K4me1, H3K4me3, H3K9me3, H3K27me3 or PU.1. For immunoprecipitation sheared chromatin of 1 million cells was added to the preincubated beads over night at 4°C. Chromatin complexes were isolated by magnetic bead selection and washed with RIPA and TE buffer. Chromatin complexes were digested with 50 µg/ml RNase (Roche) at 37°C for 30 min. Immunoprecipitated DNA was purified using QIAquick PCR Purification Kit according to the manufacturer's protocol (Qiagen). DNA concentration of immunoprecipitated DNA was determined by using Qubit dsDNA HS Assay kit (Life Technologies). Libraries were prepared and subjected to deep-sequencing on the Illunima platform according to then manufacturer's protocols.
GEO Accession
GSM1384944