Alternative mRNA isoforms identified by RNA-seq analysis during the critical transition of terminal erythroid differentiation. (A) Distribution of the number of significant isoform events per genes comparing R2 with all following stages of erythropoiesis. (B) Pie chart showing the proportion of isoform changes affecting amino acid sequences in the 6 different classes of gene isoform expression events. (C) Statistically significant GO terms of genes with protein coding changes due to alternative isoforms. (D) Sashimi plots⁵³  of the events showing exonic reads and junctional reads of the exon trios of the indicated SE events from (top to bottom) R2 to R5. The height of the wiggles in the exons depicts the log2 RPKM of reads covering those positions. Arcs connecting exon boundaries represent junctional reads that align the 2 connecting exons. The thickness of the arcs is proportional to the number of corresponding junctional reads. (E) Semiquantitative isoform RT-PCR analysis of splicing events in R2 proerythroblasts and late erythroblasts (R3 and R4) isolated from fetal livers at E14.5. Arrows mark the inclusion product (top band) relative to the exon-skipping product (bottom band). The bar graphs below the gel images show percentages of the included isoform (Ψ value) as quantified from the relative intensities of the inclusion and exclusion bands. (F) Quantitative RT-PCR determination of the absolute levels of expression of the inclusion and exclusion isoforms. **Significance (P < .01). N.S., nonsignificant.