Fig. 7: INSCRIBE unveiled RNA targets of RBFOX2 in mouse brain tissue. | Nature Communications

Fig. 7: INSCRIBE unveiled RNA targets of RBFOX2 in mouse brain tissue.

From: High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues

Fig. 7

a Schematics of RBFOX2-INSCRIBE with mouse brain tissue. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. b HOMER de novo motif discovery identified the canonical RBFOX2 motif UGCAUG as the top motif in MeOH-fixed and PFA-fixed mouse brain INSCRIBE confident edit clusters, but not the enzyme-only control, using a cumulative hypergeometric distribution for p values. c A density plot of the distribution of the closest UGCAUG distance to confident edit clusters for INSCRIBE in MeOH-fixed mouse brain tissue (orange), PFA-fixed mouse brain tissue (green) and both enzyme-only controls (blue and purple). A peak at 0nt distance indicated the enrichment of UGCAUG in proximity to the edit cluster center. d, e The actual fraction of UGCAUG-containing mouse brain RBFOX2-INSCRIBE edit clusters (dots) was significantly higher than that of the 20 permuted clusters (box plot, n = 20). Z-scores: RBFOX2-INSCRIBE (Mouse Brain MeOH-fixed), 30.81; RBFOX2-INSCRIBE (Mouse Brain PFA-fixed), 28.05; Enzyme-only control (Mouse Brain MeOH-fixed), 1.95; Enzyme-only control (Mouse Brain PFA-fixed), 1.30. e, The actual fraction of mouse brain eCLIP-overlapping mouse brain RBFOX2-INSCRIBE edit clusters (diamonds) was significantly higher than that of the 20 permuted clusters (box plot, n = 20) but not the enzyme-only control, demonstrating edit clusters specifically captured mouse RBFOX2 targeted RNA sites. The eCLIP experiment was performed in mouse whole brain and the IDR peaks were used for the comparison. Z-scores: RBFOX2-INSCRIBE (Mouse Brain MeOH-fixed), 37.75; RBFOX2-INSCRIBE (Mouse Brain PFA-fixed), 40.89; Enzyme-only control (Mouse Brain MeOH-fixed), 7.65; Enzyme-only control (Mouse Brain PFA-fixed), 6.06. In (d, e), the actual fraction was plotted using the confident edit clusters derived from 3 INSCRIBE technical replicates. Enrichment was defined as the ratio of actual fraction to the mean of the permuted-derived fraction. The box shows the quartiles while the whiskers extend to show the rest of the distribution; the median is represented by the center line. f Integrative genome viewer (IGV) tracks of example mouse RBFOX2 target genes (Ppp3ca, Klc1) with 1500 bp windows showing mouse RBFOX2 eCLIP peaks (orange), MeOH-fixed and PFA-fixed mouse brain RBFOX2-INSCRIBE (blue) and the respective enzyme-only controls (gray). The overlay displays the edit fraction quantified by SAILOR in 3 replicates of INSCRIBE, alongside with the confident edit clusters identified by FLARE.

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