Sybr green fluorescent dye

Cells were lysed with RIP buffer [150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5 mM DTT, 0.5% NP40 and 100 U/ml RNase inhibitor (Enzynomics, Daejeon, Korea)] containing a protease inhibitor cocktail (Roche Applied Science). Cell lysates containing 600 μg of protein were incubated with the anti-RBFOX2 antibody (Bethyl Laboratories) or control rabbit IgG (Sigma-Aldrich) for 4 h at 4°C, and the lysate was centrifuged at 10,000 × g for 10 min. Dynabeads^® Protein G (50 μl; Invitrogen, CA, USA) was washed three times with RIP buffer and incubated with the cell lysate for 2 h at 4°C, after which the beads were washed six times with RIP buffer. Total RNA was extracted using a Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA, USA) and reverse-transcribed using M-MLV reverse transcriptase (Promega) with random hexamers. The primer set for the TEAD1 and TEAD2 intronic region used in this experiment is shown in Supplementary Table S3. RIP-qPCR was performed using SYBR Green fluorescent dye (GENET BIO) and the AriaMx PCR system (Agilent Technologies). Normalization was performed using β-actin as an internal control.

Total RNA was extracted using a Hybrid-R RNA extraction kit (GeneAll, Seoul, Korea) according to the manufacturer's instructions and reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA) with random hexamers. qRT-PCR was performed using SYBR Green fluorescent dye (GENET BIO, Daejeon, Korea) and the AriaMx PCR system (Agilent Technologies, Santa Clara, CA, USA). Normalization was performed using β-actin as an internal control. RT-PCR was performed with GoldHotStart Taq PCR master mix (Bioneer) using a SimpliAmp thermal cycler PCR machine (Applied Biosystems, Waltham, MA, USA). The specificity of each PCR product was assessed by agarose gel electrophoresis, and band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA). The ratio of TEAD1 transcripts derived from AS was calculated using the following formula: where N is the skipped-isoform product size/inclusion isoform product size. The primer set used in this experiment is shown in Supplementary Table S2.