Fig. 1

(A) Western blot analysis of HeLa cell lysates corresponding to 2×10⁵ cell equivalents of cells transiently transfected with the indicated knockdown constructs. Membrane sections were incubated with antibodies against UPF1, SMG6, SMG7, and Tyr-Tubulin, the latter serving as a loading control. (B) Upper part: Schematic representation of how long and short-read sequencing are combined to identify endogenous NMD-sensitive mRNA isoforms in human cells. Boxes denote exons (NMD-inducing exons in blue,), green lines denote long and short sequencing reads, long purple lines denote long reads that correspond to NMD-sensitive isoforms, short blue lines denote short reads that map to exons of NMD-sensitive isoforms. Lower part: Representation of the short-reads expression level patterns of NMD-sensitive exons. NMD-sensitive isoforms can occur by exon inclusion or exon exclusion and the patterns of changes of the expression levels to opposite directions are taken into consideration. (C) Bar plot of the number of genes and transcripts detected by long-read cDNA sequencing in different experimental conditions. (D) Histogram depicting the number of isoforms that were detected per gene cumulated over all the conditions. (E) Schematic illustration of the bioinformatics pipeline for analysing NMD-sensitive mRNA isoforms using long and short-read data. The components of the pipeline shaded in light blue describe input/output files and the boxes in red represent the computational tools that were applied