Extended Data Fig. 5: TDP-43 eCLIP on skeletal muscle myoblasts and myotubes. | Nature

Extended Data Fig. 5: TDP-43 eCLIP on skeletal muscle myoblasts and myotubes.

From: TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle

Extended Data Fig. 5

Related to Fig. 3. a, RNA immunoprecipitation of C2C12 myotubes, followed by oligo-dT northern blot. Analyses reveal that A11 and TDP-43 associate with poly-A RNA. n = 3 biologically independent samples. b, Schematic of the eCLIP protocol for cultured C2C12 myoblasts and myotubes. c, Immunoprecipitation of TDP-43 complexes used for eCLIP in C2C12 myoblasts. n = 2 biologically independent samples. d, Same as in c, but for C2C12 myotubes. n = 2 biologically independent samples. e, Autoradiogram of 32P-labelled TDP-43–RNA complexes fractionated by PAGE. White boxes indicate the area cut and used for eCLIP library preparation. n = 1 library was prepared per condition. f, Top, scatter plots indicate correlation between significant TDP-43 eCLIP peaks in biological replicates. Scatter plots represent fold enrichment for each region in TDP-43 eCLIP relative to paired size-matched input with significant peaks in red (P ≤ 10−8 over size-matched input). P values for each peak to determine significance were calculated by Yates’ χ2 test (Perl), or Fisher exact test (R computing software) when the expected or observed read number was below five16. For myoblasts, R values were calculated using n = 511,137 non-significant peaks and n = 596 significant peaks. For myotubes, R values were calculated using n = 413,368 non-significant peaks and n = 1,501 significant peaks. Bottom, the UG-rich motif is significantly enriched in clusters from open reading frames and untranslated regions (UTRs). E values were determined using the DREME software tool. g, Irreproducible discovery rate analysis comparing peak fold enrichment across indicated datasets. h, TDP-43 eCLIP reveals that TDP-43 binds to the 3′ UTR of the TDP-43 transcript in myoblasts (top) and myotubes (bottom). n = 3 biologically independent experiments, each showing similar results.

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