Figure 4. Splicing order is consistent between alternative isoforms and displays later removal of AS introns.

A) Top: dRNA-seq coverage tracks of one representative biological replicate for two example alternative cassette exons (yellow) with differential inclusion during sMN differentiation. AS introns are shown in yellow font throughout the figure. Bottom: Validation of AS events by RT-PCR of total RNA. The region amplified by PCR is indicated below each gel. NO-RT: no reverse transcriptase control. B) Splicing order plots for two intron groups that contain AS introns, shown separately for inclusion and exclusion of the alternative exon and for each timepoint that had sufficient coverage for a given isoform. C) Left: Heatmap displaying hierarchical clustering of inclusion levels from short-read RNA-seq for exons that are differentially included between Days 9 and 14 and for which splicing order was computed. Right: Heatmap displaying the summed splicing order scores of AS introns for exclusion and inclusion isoforms at each timepoint. Rows represent intron groups and are ordered according to the exon inclusion heatmap. N/A indicate orders that were not observed for an intron group or for which splicing order could not be computed because of low coverage. D) Distribution of summed splicing order scores for all the possible splicing orders of AS introns shown in C). Boxplots elements are shown as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, intron groups. E) Schematic of human myoblasts differentiation into myotubes. F) Left: Schematic representation of TPM2 isoforms with mutually exclusive exons 6A and 6B. Right: Proportion of reads mapping to each TPM2 isoform in cDNA-PCR nanopore sequencing of chromatin-associated RNA from undifferentiated myoblasts and myotubes. G) Splicing order plots for the two TPM2 alternative isoforms in myoblasts and the only isoform expressed in myotubes.