GSM3656533: ChIPseq-WT-Epi-Jun_rep2; Mus musculus; ChIP-Seq
ChIP was performed as described previously(Liu et al., 2015), Briefly, cells were fixed in 18 ml DMEM (Hyclone) containing 1% formaldehyde for 15 min at room temperature with rotation, the reaction was quenched by the addition of 2 ml of 0.125 M glycine. And then the cells were washed with PBS 3 times. Cells were lysed in ChIP buffer A (50mM HEPES-KOH, 140 mM NaCl, 1mM EDTA (pH 8.0), 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 50 mM Tris-HCl (pH 8.0), and protease inhibitor cocktail) for 10 min at 4°C. Pellets were lysed in ChIP buffer B (1% SDS, 50 nM Tris-HCl (pH 8.0), 10mM EDTA and protease inhibitor cocktail) for 5 min at 4°C. The DNA was fragmented to 100-500 bp by sonication, and then centrifuged at 12,000g for 2 min. The supernatant was diluted with ChIP IP buffer (0.01% SDS, 1% Triton X-100, 2mM EDTA, 50mM Tris-HCl (pH 8.0), 150 mM NaCl and protease inhibitor cocktail). Immunoprecipitation was performed using 2μg of H3K27ac antibody added to protein A/G Dynabeads, and incubated overnight at 4°C. Beads were washed, eluted and reverse crosslinked. DNA was purified by using the MinElute Reaction Clean up Kit (QIAGEN). The ChIP DNA library for NextSeq 500 sequencing was constructed with VAHTS Turbo DNA Library Prep Kit for Illumina (Vazyme Biotech) according to manufacturer's instructions. AMPure XP beads were used for purification steps. The library was quantified with VAHTS Library Quantification Kit for Illumina (Vazyme Biotech). Libraries were sequenced on an Illumina NextSeq 500 v2 using 50bp paired-end reads.
GEO Accession
GSM3656533