Figure 2.

PA-m⁶A-seq applied to poly(A)-tailed RNA purified from HeLa cells. In following figures, blue bars represent methylation sites identified by PA-m⁶A-seq, whereas blue ‘peaks’ above those bars are from normal m⁶A-seq. a) Validation of PA-m⁶A-seq strategy. Metagene profile and pie-chart of the enrichment of RNA segments are consistent with previous reported distribution of m⁶A, and the motif search yielded GGACU as the predominant one, which was the same as the result from normal m⁶A-seq. b) Comparison of predicted methylation sites in β-actin (ACTB) and homo sapien basigin (BSG) from PA-m⁶A-seq with peaks from normal m⁶A-seq and single sites by SCARLET. Sequences of predicted sites are shown below, consensus motif containing m⁶A in red. All input background of normal m⁶A-seq has been subtracted. Both sites were verified by SCARLET. c) Multiple methylation sites in MALAT1 transcript. The methylation sites confirmed by SCARLET, which were covered by peaks from normal m⁶A-seq, were also identified by PA-m⁶A-seq with higher resolution. Yellow regions indicate the RRACH motif. Red lines are the probes used to verify these sites with SCARLET. d) Distance of predicted methylation sites using PA-m⁶A-seq versus peaks obtained from normal m⁶A-seq to YTHDF2-binding sites. e) Distance of predicted methylation sites obtained from PA-m⁶A-seq versus peaks obtained from normal m⁶A-seq to HuR-binding sites.