Fig. 6: INSCRIBE exhibited comparable performance in PFA-fixed cells. | Nature Communications

Fig. 6: INSCRIBE exhibited comparable performance in PFA-fixed cells.

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

Fig. 6

a HOMER de novo motif analysis of confident edit clusters revealed the canonical RBFOX2 motif UGCAUG (top motif) or TDP-43 motif UGUGUG (2nd top motif), for RBFOX2-INSCRIBE or TDP-43-INSCRIBE in paraformaldehyde (PFA)-fixed HEK293T cells respectively, using a cumulative hypergeometric distribution for p values. b A density plot of the distance between edit clusters to closest UGCAUG (for RBFOX2-INSCRIBE, upper panel) or UGUGUG (for TDP-43-INSCRIBE, lower panel). INSCRIBE in MeOH-fixed and PFA-fixed cells presented no obvious distinction in canonical motif enrichment at the INSCRIBE confident edit cluster center. c, d Actual fraction of motif-containing INSCRIBE edit clusters (dots) compared with that of the 20 permuted clusters (box plot, n = 20). Top: RBFOX2-INSCRIBE in PFA-fixed HEK293T cells. Z-scores: RBFOX2-INSCRIBE (PFA-fixed), 29.27; Enzyme-only control (PFA-fixed), 0.54; RBFOX2-STAMP: 23.08; Bottom: TDP-43-INSCRIBE in PFA-fixed HEK293T cells. Z-scores: TDP-43-INSCRIBE (PFA-fixed), 4.61; Enzyme-only control (PFA-fixed), −1.02. d The actual fraction of eCLIP-overlapping INSCRIBE edit clusters (diamonds) was significantly higher than that of the 20 permuted clusters (box plot, n = 20). Top: RBFOX2-INSCRIBE in PFA-fixed cells. Z-scores: RBFOX2-INSCRIBE (PFA-fixed), 18.67; Enzyme-only control (PFA-fixed), 1.36; RBFOX2-STAMP: 5.45; Bottom: TDP-43-INSCRIBE in PFA-fixed cells. Z-scores: TDP-43-INSCRIBE (PFA-fixed), 7.10; Enzyme-only control (PFA-fixed), −0.62. The actual fraction was plotted using the confident edit clusters derived from 3 INSCRIBE technical replicates. The box shows the quartiles while the whiskers extend to show the rest of the distribution; the median is represented by the center line. e Integrative genome viewer (IGV) tracks of example RBFOX2 target gene (PKM, OGT) with 1500 bp windows showing eCLIP peaks (orange), INSCRIBE in PFA-fixed cells (blue) and the enzyme-only control (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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