Chromatin immunoprecipitation (ChIP)

ES-2 wild-type and S1PR1-deleted cells were fixed in 1% formaldehyde for 10 minutes and quenched with 0.125 M glycine for 5 minutes at room temperature (18 °C). Harvested cells were washed with ice-cold PBS and then lysed in ice-cold PBS containing protease inhibitor. The mixture was centrifuged at 2,000 × g for 5 minutes at 4 °C to pellet the nuclei, and the chromatin DNA was digested with micrococcal nuclease (MNase) in ChIP MNase buffer. Immunoprecipitation reactions were carried out with chromatin extracts overnight at 4 °C using 2 μg of antibodies against YAP. Histone H3 was used as the positive control and rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) was used as the negative control. Two percent of the chromatin extract was set aside for input. Antibody-protein-DNA complexes were bound to 30 μL of ChIP-grade protein G magnetic beads (Cell Signaling Technology) for 2 hours at 4 °C. The beads were washed 3 times with low-salt wash buffer and once with high-salt wash buffer. Beads were resuspended in elution buffer and incubated for 30 minutes at 65 °C with frequent shaking. The resulting eluate and input samples were transferred into new tubes and reverse cross-linked by adding 6 μL of 5 M NaCl and 2 μL of proteinase K, and incubated for 2 hours at 65 °C. The DNA was purified using DNA spin columns. Precipitated DNA was quantitated by real-time quantitative PCR (RT-PCR) according to the manufacturer’s instructions (Simple ChIP Enzymatic Chromatin IP Kit, Cell Signaling Technology). The primers are presented in Table S1.4.