Brightgreen express 2 qpcr mastermix

For ChIP analyses, SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling) was used as described by the manufacturer. Briefly, vCTBs were transfected with YAP-5SA and control plasmids, seeded onto fibronectin-coated dishes, and incubated for 20 h. Next, cells were fixed with 1% formaldehyde (10 min). After nuclei preparation, chromatin digestion, and sonication, purified chromatin lysates were incubated either with YAP, TEAD4, EZH2, H3K27me3 (SI Appendix, Table S1), or normal rabbit IgG (negative control) overnight at 4 °C. Immunoprecipitated chromatin was captured with ChIP-Grade Protein G Magnetic Beads and eluted. Purified DNA was assessed by qPCR (7500 Fast Real-time PCR system) using ABI BrightGreen Express 2× qPCR MasterMix (ABM) according to the manufacturer’s instruction. Primers amplifying genomic regions with TEAD4 binding sites are indicated in SI Appendix, Table S2. Relative occupancy of YAP, TEAD4, EZH2, and H3K27me3 was normalized to normal rabbit IgG.

Total RNA was isolated with the TRIzol reagent (Takara, Dalian, China) and reverse transcribed using PrimeScript RT reagent Kit (Takara, Dalian, China). Quantitative real-time PCR was used in the BrightGreen Express 2 × qPCR MasterMix (Abm, Canada). The relative gene expression levels were normalized to GAPDH and calculated by means of the 2^−ΔΔCT method.