FIGURE 3.

In vitro DART-seq identifies m⁶A transcriptome-wide. (A) Comparison of C-to-U editing rates in methylated mRNAs obtained by in vitro DART-seq and cellular DART-seq. Sanger sequencing traces show C-to-U editing adjacent to m⁶A sites in a panel of five methylated mRNAs: DDX5, TUB, EIF4B, MKLN1, and HERC2. m⁶A sites are indicated by asterisks. C-to-U editing rate (%U) is indicated above the adjacent cytidine. Data Representative of three biological replicates. (B) Genome browser tracks of in vitro DART-seq data showing C-to-U editing in three representative mRNAs: ZZZ3, ATRX, and EEF1A1. C-to-U editing found in at least 10% of the reads is indicated by green/yellow coloring. m⁶A peaks identified by MeRIP (Meyer et al., 2012) is indicated in the bottom blue track. (C) Metagene analysis of m⁶A sites identified by in vitro DART-seq using the APO1-YTH^D422N protein. (D) Venn diagram showing the overlap between methylated RNAs identified by in vitro DART-seq filtered against the APO1-YTH^mut negative control and methylated RNAs identified by antibody-based methods (Meyer et al., 2012; Schwartz et al., 2014; Lichinchi et al., 2016). (E) Absolute distance plot showing the distance of C-to-U editing sites identified by in vitro DART-seq relative to m⁶A sites identified by miCLIP (Linder et al., 2015). m⁶A sites are centered at position 0. (F) Venn diagram showing the overlap between methylated RNAs identified by in vitro DART-seq compared to methylated RNAs found by cellular DART-seq.