Instrument: NextSeq 550
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Protein complexes were cross-linked with 1.5 mM ethylene glycol bis(succinimidyl succinate) (21565; Thermo Scientific) in PBS with gentle agitation at RT for 15 minutes. Cross-linking of protein complexes to DNA was performed by adding methanol-free formaldehyde to a final concentration of 1% and incubation with gentle agitation at RT for 8 minutes. Next, cross-linking was quenched by adding glycine to a final concentration of 200 mM at RT for 5 minutes. Cells were then washed three times with ice-cold PBS containing 1% protease inhibitor cocktail, collected by scraping, and pelleted by centrifugation at 2000 × g for 5 minutes at 4°C. Nuclei were purified by re-suspending pellets in buffer LB1 (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1% protease inhibitor cocktail) and washed in buffer LB2 (10 mM Tris HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% protease inhibitor cocktail) followed by lysis in buffer LB3 (10 mM Tris HCl p.H 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 1% NP-40, 1% Triton X-100, 1% protease inhibitor cocktail) while rotating for 1 hour at 4°C. Chromatin samples were sheared twice using a BioRuptor PLUS device (B01020001; Diagenode, Liège, Belgium) for 6 minutes at 4°C. Correct chromatin fragment size was verified using gel electrophoresis. Dynabeads (10002D; Thermo Fisher Scientific) were incubated for 4 hours with primary antibodies. YAP bound chromatin was precipitated in RIPA buffer (50 mM HEPES pH 7.6, 1 mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5 M LiCL, 1% protease inhibitor cocktail) at 4°C on a rotating platform for 18 hours. Afterwards the beads were washed five times in RIPA buffer, and once with TE buffer (10 mM Tris, 1mM EDTA, pH 8.0). Immunoprecipitated chromatin was eluted with elution buffer (1% SDS, 0.1M NaHCO3), de-crosslinked overnight, and cleaned up using RNAse and Protease-K treatment, followed by purification with a PCR cleanup kit (Qiagen). Sequencing libraries were prepared for input control and two biological replicates for YAP in each culture condition using the NEBNext Ultra II DNA Library Prep Kit according to manufacturer's instructions (E7370; New England Biolabs, USA). Library quality was verified using BioAnalzyer.