Supplemental data for differential expression and splicing analysis. (A) Scatter plot of mRNA abundance for (left panel) XCR1-edited versus SUGP1-edited U-2 OS cells and (right panel) NTC versus XCR1-edited U-2 OS cells (mean log₂ normalized values from three independent experiments). mRNAs meeting threshold for inclusion (minimum absolute log₂ normalized fold change ≥0.5 and FDR ≤ 0.05) are labeled in blue, except (left panel) SUGP1, DYNC1I2, and LIS1, and (right panel) XIRP1 (the only differentially expressed gene in the NTC versus crXCR1 comparison), which are labeled in yellow. Inset tables show non-logarithmic values for (left panel) SUGP1, DYNC1I2, and LIS1 and (right panel) XIRP1 mRNAs. See Table S9 for full results. (B) Venn diagram showing overlap of differentially expressed genes in the NTC versus crSUGP1 and crXCR1 versus crSUGP1 comparisons. (C) Quantification of LIS1 and DYNC1I2 mRNA level, determined by TaqMan-based real-time qPCR, in SUGP1-edited and XCR1-edited U-2 OS and ARPE-19 cells. Data points represent the mean of three independent experiments (RQ = relative quantification based on NTC). Error bars signify SD. *P < 0.05, **P < 0.01 (one-way ANOVA with Dunnett’s multiple comparison against NTC). (D and E) Venn diagrams showing the overlap of genes that undergo differential splicing (D) and differential splicing events (E) in the datasets, as determined with rMATs (note that some genes have >1 differential splicing event). The threshold for classifying an event as differential was: absolute IncLevelDifference ≥0.2, total read count (inclusion count + skipping count) ≥10, and FDR ≤ 0.05. See Table S10 for full results. (F) Classes of alternative splicing events common to both comparisons (i.e., NTC versus crSUGP1 or crXCR1 versus crSUGP1), as identified by rMATS. Blue and green represent events that were enriched in control and crSUGP1 samples, respectively. rMATS reports 5 splicing categories: (i) alternative 3′ splice sites (A3SS); (ii) alternative 5′ splice sites (A5SS); (iii) mutually exclusive exons (MXE); (iv) retained introns (RI), and (v) skipped exons (SE). The A3SS and A5SS events involve the splicing together of two exons separated by a single intron. For A3SS events, alternative splicing causes a downstream exon to extend partially into neighboring intronic sequence. A5SS is defined by an alternative splicing event causing an upstream exon to extend partially into the adjoining intron. MXEs describe the splicing of adjacent exons (separated by a single intron) in which one exon is retained but the other is excluded, or vice versa. The graph reports MXE events in which the upstream exon was selected. Events classified as RI are those in which an intron is not spliced out and hence is retained in the mature transcript. SE denotes splicing events in which an exon is skipped over and not included in the processed RNA molecule. (G) Venn diagrams showing overlap of differential 3′-end usage events in the datasets, as determined by LABRAT. LABRAT quantifies alternative polyadenylation sites and reports upstream or downstream shifts in the usage of those sites for each gene as compared to the control (see Table S11 for full results). The threshold for classifying an event as differential was Δψ ≥0.05 and FDR ≤ 0.05.