Anti yap1 14074s

In total, ~1 million cells were cross linked with 1% formaldehyde at room temperature for 10 min, then double cross-linked with 2 mM DSG (ProteoChem, Hurricane, UT, YSA) for 1 h, followed by another 10 min with 1% formaldehyde. Cross-linking was quenched with 0.125 M glycine for 5 min. After cell lysis and nuclear extraction, chromatin was sonicated in lysis buffer with Q800R sonicator (QSonica, Newtown, CT, USA) to acquire ~200–500 bp fragments. Soluble chromatin was incubated with 3–6 mg of antibody at 4 C overnight. The antibodies used in ChIP included anti-YAP1 (14074S, Cell Signaling Technology, Danvers, MA, USA), anti-SMAD4 (38454S, Cell Signaling Technology), and anti-H3K27ac (ab4729, Abcam, Waltham, MA, USA). Immunoprecipitated complexes were pulled down using 50 µL Dynabeads Protein G (Life Technologies, Carlsbad, CA, USA). Beads were washed with washing buffer (50 mM HEPES pH7.6, 1 mM EDTA, 0.7% Na Deoxycholate, 1% NP-40, 0.5 M LiCl) six times to minimize the background. Protein-DNA complexes were then eluted, and DNA fragments were purified. Sequencing was performed using the Illumina’s HiSeq 3000 System.