Fig. 5: scCHyMErA-Seq identifies cassette exons influencing cell cycle distribution.

a Stacked bar plot summarizing exons whose deletion affects cell cycle phase distribution, as identified by scCHyMErA-Seq. Phases with significantly increased cell fractions are indicated (****p < 0.0001, ***p < 0.001, **p < 0.01; One-sided Fisher’s exact test with Benjamini–Hochberg correction). b RT-PCR analysis of exon inclusion for the indicated genes in RNA from HAP1 cells transduced with two independent intergenic control hgRNAs or two independent hgRNAs targeting the indicated exons using scCHyMErA-seq–compatible constructs. Single experiment performed. c Validation of scCHyMErA-Seq results by propidium iodide staining and flow cytometry. Stacked bar plots indicate the effect of exon deletions on cell cycle distribution; significantly increased phases are indicated. Data represent mean ± SD from three biological replicates, each calculated as the average of two independent hgRNA treatments; **p < 0.01, *p < 0.05; one-way ANOVA. d Scatter plot comparing log2 fold-change values of differentially expressed genes across all perturbations between gene knockouts and exon deletions. The two-sided Pearson’s correlation coefficient is shown. e Volcano plot of DESeq2 pseudobulk analysis comparing SKP2 exon-6 deletion with SKP2 knockout. Significantly upregulated (red) and downregulated (blue) genes are indicated (adjusted p < 0.05, |log2 fold-change|> 0.5). p values were computed using DESeq2’s Wald test with Benjamini–Hochberg correction. f Scatter plot comparing log2 fold-change values of genes between SKP2 knockout versus intergenic controls (x-axis) and SKP2 exon-6 deletion versus intergenic controls (y-axis). Only genes differentially expressed between SKP2 knockout and exon-6 deletion are shown. g KEGG pathway enrichment analysis of differentially expressed genes between SKP2 knockout and SKP2 exon-6 deletion. Pathways with padj <0.01 (****p < 0.0001, ***p < 0.001; Fisher’s exact one-tailed test with multiple-testing correction using g:Profiler’s g:SCS method); all expressed genes were used as background.