Fig. 4

Classification and validation of the novel transcripts identified by Iso-seq. a Pie chart showing percentages of novel transcripts with single or multiple exons, with or without detectable alternative splicing events. b Pie chart showing percentage of novel transcripts from the antisense strand of annotated genes. c Pie chart showing novel transcripts that are likely non-coding or with coding potentials. Those with coding potential scores (> 0) calculated by Coding Potential Calculator 2 software were considered coding. d, e RT-PCR validation of novel transcripts with coding potential (d) and non-coding novel transcripts (e). The left panels show diagrams of selected transcripts. White boxes, gray boxes, and lines represent the untranslated regions, CDS, and introns, respectively. Forward and reverse primers for PCR are showed as blue arrowheads. Right panels: gel images showing amplified RT-PCR products from D4h (D) and L4h (L) cDNAs. RT and -RT, reverse transcription to produce cDNA with or without reverse transcriptase, respectively. UBQ 5 was a RT-PCR control