GSM6621039: uninduced MCF10A YAP5SA cells p63; Homo sapiens; ChIP-Seq
For ChIP-seq, cells were cross-linked with 1% formaldehyde (Sigma) for 10 min at room temperature. The reaction was stopped by adding 125 mM glycine (Sigma). After cells were lysed for 10 minutes on ice [5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP40, 1 mM PMSF, protease inhibitor cocktail (Sigma)], nuclei were resuspended in RIPA buffer [50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, protease inhibitor cocktail (Sigma)]. Chromatin was fragmented to an approximate length of 150 to 300 bp using a Branson sonifier. Antibodies (9 µg) were coupled to protein G dynabeads (Thermo Fisher Scientific) for 6 hours at 4°C and then incubated with fragmented chromatin over night at 4°C. Beads were washed in total twelve times with wash buffer I (50mM Tris-HCl pH8, 0.15M NaCl, 1mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), wash buffer II (50mM Tris-HCl pH8, 0.5M NaCl, 1mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), wash buffer III (50mM Tris-HCl pH8, 0.5M LiCl2, 1mM EDTA, 1% Nonidet P-40, 0.7% sodium deoxycholate) and wash buffer IV (10mM Tris-HCl pH8, 1mM EDTA). 1mM PMSF and protease inhibitor cocktail (Sigma ) was added freshly to all buffers. Chromatin was eluted in (10mM Tris-HCl pH8, 0.3M NaCl, 5mM EDTA, 0.5% SDS, 10µg/ml RNaseA) and crosslink was reversed at 65°C over night. Proteins were digested by adding 200 µg/ml proteinase K at 55°C for 2 hours. DNA was purified using the QIAquick PCR Purification Kit (QIAGEN) and eluted in 50 µl EB buffer. For ChIPseq, DNA libraries were generated using 10ng purified ChIP-DNA and the NEBNext®Ultra II DNA Library Prep Kit for Illumina® (New England Biolabs) according to the manufacturer's instructions. DNA library was amplified by 15-18 PCR cycles and quality was analyzed using the Fragment Analyzer (Advanced Analytical). Libraries were sequenced on the NextSeq 500 platform (Illumina).