GSM1887717: CDP_H3K27ac_1; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed as described before (Dahl and Collas, 2008; Chauvistré et al., Eur. J. Immunol. 44, 2478-2488, 2014) 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 in Bioruptor with cooling device (Diagenode) at 4°C for 10 min with 30 s pulse/pause cycles. Sheared lysates were clarified by centrifugation at 12,000g (10 min, 4°C). 10 µl Dynabeads Protein A (Life Technologies) were preincubated with either 1 µg H3K27Ac or IgG control (both Santa Cruz Biotechnology). 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 (Qiagen). DNA concentration of immunoprecipitated DNA was determined by using Qubit dsDNA HS Assay kit (Life Technologies). RNA was isolated using RNeasy Mini Kit with DNaseI digestion (Qiagen) as before (Felker et al., J. Immunol. 185, 5326-5335, 2010) ChIP-Seq Libraries were prepared and subjected to deep-sequencing on the Illunima platform according to then manufacturer's protocols. RNA-Seq libraries were prepared and subjected to strand-specific RNA-seq on the Illumina platform according to the manufacturer's protocols.
GEO Accession
GSM1887717