Semi‐quantitative and quantitative reverse transcriptase (RT)–PCR

Reverse transcription was performed with 1–4 µg of total RNA in a 20 µl reaction volume with 10 µM VNN‐(dT)₂₀ primer and the GoScript Reverse Transcriptase (Promega). For the semi‐quantitative end‐point PCRs, the MyTaq Red Mix (Bioline) was used. Quantitative RT–PCRs were performed with the GoTaq qPCR Master Mix (Promega), 2% of cDNA per reaction, and the CFX96 Touch Real‐Time PCR Detection System (Bio‐Rad). Each biological replicate was repeated in technical triplicates and the average Ct (threshold cycle) value was measured. When isoform switches were measured, values for NMD sensitive isoforms were normalized to the canonical isoforms to calculate ΔCt. For differentially expressed genes, the housekeeping gene C1orf43 or EMC7 values were subtracted from the target value to receive the ΔCt. To calculate the mean log2 fold changes, three biologically independent experiments were used. The log2 fold changes are visualized as single data points and mean. All primers used in this study are listed in Dataset EV7.