Alternative splicing validation by RT-PCR

RT reactions were performed using the High Capacity cDNA RT Kit (Applied Biosystem #4368814) and PCRs using GoTaq DNA Polymerase (Promega #M7123). In both cases, manufacturer protocols were followed. PCR program contained the following steps: (i) 95 °C for 1 min 45 s, (ii) 28 cycles of 95 °C for 45 s, 57 °C for 45 s and 72 °C for 1 min, (iii) 72 °C for 10 min, and (iv) 25 °C for 5 min. Primer sequences (Sigma) for the alternative splicing events evaluated are described in Table S8. PCR products were analyzed by 6% PAGE. We quantified the percentage spliced in (psi)³⁷ of the alternative regions by densitometry using ImageJ plugin for gel analysis and following equation 1 (Eqn 1).