GSM1581522: CREB; Mus musculus; ChIP-Seq
Aliquots of 5x106 cells were lysed (10 min 4 °C) in lysis buffer containing 50 mM Tris-HCl pH 8.1, 10 mM EDTA and 1 % SDS, supplemented with Protease Inhibitor Cocktail® (SIGMA-ALDRICH, Gillingham, UK) and Phosphatase Inhibitor Cocktail Set II® (Calbiochem, Watford, UK), then diluted (2:3) in a buffer containing 20 mM Tris-HCl, 150 mM NaCl pH 8.1, 1 % Triton X-100 and 1 mM EDTA. Lysates were sonicated for 45 min on ice using a Bioruptor plus® (Diagenode, Seraign, Belgium) 30s ON and 30s OFF at HIGH setting. Sonicated cellular lysate was centrifuged (15000 g 10 min 4°C), supernatants were pre-cleared with 30μl of Protein A Agarose/Salmon sperm DNA (50 % slurry) (Merk Millipore, Watford, UK) for 30 min at 4 °C with agitation. Agarose was removed by brief centrifugation (500 g 4 °C 1 min) and supernatants were incubated overnight with gentle agitation at 4 °C with anti-CREB (clone 48H2) (Cell Signaling Technology, Leiden, The Netherlands). Samples were then incubated with 30 μl of Protein A Agarose/Salmon sperm DNA (50 % slurry) for 1 hour at 4 °C with gentle agitation. The agarose pellet with bound DNA was recovered and washed for 5 min each with 1ml each of low salt buffer, then high Salt buffer, then LiCl buffer and finally twice with TE buffer (all Merk Millipore, Watford, UK). DNA was recovered by incubating agarose pellets for 2 hours at 68 ºC in an elution buffer containing 20 mM Tris–HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS and 50 μg/ml proteinase K (Promega, Madison, USA) with vigorous shaking. Agarose was removed by gentle centrifugation and DNA extracted from supernatants using phenol-chloroform. DNA libraries, sequencing, peak calling, and enrichment analysis were carried out by the Centre for Genomic Research at the University of Liverpool, Liverpool, UK. Briefly, DNA for each sample was end repaired, A-tailed and ligated to an appropriate dilution of Illumina adapter. The ligated product was then amplified to generate the final library, which were pooled in equimolar ratios using Qubit and Bioanalyzer data, and quantified by qPCR, using an Illumina Library Quantification Kit (KAPA, KK4854) on a Roche LightCycler 480II system.  Template DNA was denatured according to the protocol described in the Illumina cBot User Guide and loaded at 8.5 pM concentration. Sequencing was carried out on 1 lane of an Illumina HiSeq 2500 with version 1 chemistry generating 2 × 100bp paired end reads
GEO Accession
GSM1581522