Fig. 3: Validation of the AS events of long-read sequencing data at both transcription and translation levels.

a Validation of splice junctions by bulk RNA-seq data. The proportions of validated and nonvalidated splicing junction motifs by RNA-seq in each category of transcripts (the number refers to the validated and nonvalidated splice motifs number). b The bar chart indicates the number of validated transcripts (all the junctions were validated) by RNA-seq. c The proportion of AS patterns varied in different cochlea cell subtypes. d, e Validation of the novel splicing isoforms of deafness-related genes using RNA-seq, reverse transcription-polymerase chain reaction (RT–PCR) and Sanger sequencing (representative results of at least three biological replicates are shown). Transcript structure and name are shown in the left panel, with protein-coding exons, noncoding exons, and introns represented by filled boxes, open boxes, and lines. The forward and reverse PCR primers are marked with lines and arrowheads in the relative transcript structure. Sashimi plot showing the sequencing read coverage for differentially spliced events and novel splice junctions are color-coded. The gel bands in each figure show standard size markers and the expected size of PCR products, and each product identity was confirmed by Sanger sequencing. f The pie chart indicates the number of transcripts supported with peptide evidence. g The unique peptide expression of each cell type after mapping the total peptides to the cell cluster ScISOr-Seq data. h Examples of the unannotated isoform-specific peptides validated by mass spectrometry-based proteomics of Coch. The upper panel represents the novel transcript, amino acid sequences (the peptide indicated in red has been detected by proteomics), and the reference transcript. The fragment ions supporting the corresponding peptide are highlighted (red, y ion; blue, b ion) in the lower panel.