Extended Data Fig. 1: UNC13A and UNC13B are misspliced after TDP-43 knockdown across neuronal lines.

(a, b) RNA-seq traces from IGV³⁴ of representative samples from control (top) and TARDBP KD (bottom) in i³Neurons showing intron retention in UNC13A (A) (mean 4.50 ± 1.50 increased IR in KD) and UNC13B (mean 1.86 ± 0.63 increased IR in KD)(B), overlaid with published TDP-43 iCLIP peaks²² (c) Histogram showing number of basescope cryptic foci per nuclei in control (blue) and TDP-43 KD (grey) in WTC11-derived i³Neurons, p < 0.0001 unpaired t-test. (d, e) RT-qPCR levels of TARDBP and UNC13A with a non-targeting control sgRNA (sgTARDBP −), an intermediate TDP-43 KD (sgTARDBP +) or a higher TDP-43 KD (sgTARDBP ++) in WTC11-derived (d) and NCRM-5-derived i³Neurons (e). n = 4 biological replicates sgTARDBP − (d), n = 6 biological replicates sgTARDBP − (e), sgTARDBP + (d, e) and ++ (d, e). plotted as means ± SEM. (f) Representative images of UNC13A CE RT-PCR products (g) Quantification of the lower gel in (f) plotted as means ± SEM, n = 6 biological replicates non-targeting control sgRNA (sgTARDBP −), sgTARDBP +, sgTARDBP ++. Upper gel is quantified in Fig. 1h. One-way ANOVA with multiple comparisons. (h–k) Expression of TDP-43 regulated splicing in UNC13A(h, i) and UNC13B(j, k) across neuronal datasets^9,21 in control (blue) and TDP-43 KD (yellow). Intron retention (IR)(i, k) and CE and fsE PSI (h, j) significantly increase after TDP-43 depletion in most experiments, Wilcoxon test (l) Relative gene expression levels for TARDBP across neuronal datasets^9,21. Normalized RNA counts are shown as relative to control mean. Numbers show log₂ fold change calculated by DESeq2. Significance shown as adjusted p-values from DESeq2. For (h–l) biological replicates are: iPSC MN Ctrl KD n = 12, TDP-43 KD n = 6; i³N Ctrl KD n = 4, TDP-43 KD n = 3; SH-SY5Y, SK-N-BE(2)^a, and SK-N-BE(2)^b Ctrl KD n = 3, TDP-43 KD n = 3, Significance levels reported as * (p < 0.05) ** (p < 0.01) *** (p < 0.001) **** (p < 0.0001).