Validation of light-regulated intron retention (IR) events. Pooled RNA from dark-grown (D), 1-h light-treated (R1 and B1), 4-h ligh-treated (R4 and B4) samples were tested in triplicate for qRT-PCR. Primer sets designed for amplifying IR regions, total transcripts of the corresponding genes, and PpACT2 were used (see Additional file 7). PpACT2 was first used as an internal control for normalization of each qRT-PCR reaction. IR level were further normalized for total transcripts, and then compared with data from the dark-grown control to generate a relative IR level. IR events were selected from red- (A) and blue- (B) light samples. To determine the IR level from RNA-seq data, the level of IR isoforms was computed for the intron reads per kilobase of retained intron per million mapped reads (IPKMs). For comparison, IPKMs were normalized with RPKMs of corresponding genes and calculated for relative IR level compared to the dark-grown control. Corresponding gene products of selected IR events and representing processes are shown in each graph and above, respectively.