GSM5597323: Yap high density; Mus musculus; ChIP-Seq
MEFs were crosslinked by adding formaldehyde to the medium to a final concentration of 1%  in 15 cm plates with continuous, gentle rocking at RT for 10 minutes. The reaction was quenched by adding 125 mM final concentration of glycine, with continuous, gentle rocking at RT for 5 minutes. The cells were washed 3 times with ice cold PBS, scraped with 10 ml of PBS, and spun down for 5 minutes at 1,500 rpm at 4°C. For Chromatin-immunoprecipitation, for cell pellets of 106 cells, 1 ml cell lysis buffer (10 mM Hepes pH 7.3, 85 mM KCl, 1 mM EDTA, 0.5% IGEPAL CA-630, 1x protease inhibitors (ThermoFisher Halt)) was used to resuspend the pellet and incubated on ice for 5 minutes. The lysate was centrifuged for 5 minutes at 4,000 rpm at 4°C, the supernatant was removed, and the pellet was resuspended in 0.3 ml of nuclear lysis buffer for sonication (10mM Tris pH 8.0, 0.5% N-lauroylsarcosine, 0.1% sodium deoxycholate, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x protease inhibitors (ThermoFisher Halt)). Cell lysate was transferred to 1.5 ml Bioruptor Plus TPX microtube (Diagenode), and was sonicated for 4 rounds of 15 cycles of 30 seconds on/30 seconds off on high power (Diagenode Biorupter plus). After sonication, the chromatin was centrifuged for 15 minutes at max speed at 4°C and the supernatant was carefully transferred to a new tube and 1% Triton X-100 was added.  0.7 mL of ChIP dilution buffer (20 mM Tris pH 7.5, 0.5% Triton X-100, 100 mM NaCl, 1 mM EDTA, 1x protease inhibitor) was then added to the sonicated chromatin.   10% of the chromatin was taken as “Input”, and the remaining chromatin incubated overnight with anti-YAP1 antibody at 1:50 dilution (CST) overnight on a rotator at 4°C. Protein G Dynabeads (ThermoFisher 10004D) were washed 3 times with PBS + 0.5% BSA and then added to the chromatin and antibody mixture and rotated at 4°C for 3 hours. Protein G dynabeads were washed sequentially with low-salt wash buffer (20 mM Tris pH 7.5, 0.1% SDS, 1 % Triton X-100, 150 mM NaCl, 1 mM EDTA), and LiCl wash buffer (10 mM Tris pH 7.5, 1% sodium deoxycholate, 1% Triton X-100, 250 mM LiCl, 1 mM EDTA) 3 times, and washed once with TE to remove detergents and salts. Bound DNA was eluted twice by resuspending the beads with 125 ul of elution buffer (50 mM NaHCO3, 1% SDS) and incubated for 10 minutes at RT, followed by 3 minutes of vigorous shaking at 37°C. Eluted DNA was digested with proteinase K at 55°C for 2 hours and reverse-crosslinked at 65°C overnight. DNA was purified using the Qiagen MinElute PCR Purification Kit and ChIPseq libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Sequencing was performed with Illumina NextSeq500 for paired-end 38bp reads.
GEO Accession
GSM5597323