Supplementary Figure 8: Characterization of antibody enrichment activity by in vitro assays and sequencing data.
From: Evolution of a reverse transcriptase to map N1-methyladenosine in human messenger RNA
a, Dot blot of m1A antibody (MBL 345–3) against short 5-mer m1A and control m6A and synthetic A oligonucleotides. b, LC–MS/MS assay for quantifying m1A level from biological RNA sample before and after immunoprecipitation with the anti-m1A antibody. Quantified percentages of m1A and m6A in reference to A are shown by mean ± s.d. of n = 3 LC–MS/MS injection replicates. c, Overlay of coverage tracks of 28S rRNA between m1A-IP-seq (‘IP’) and m1A-quant-seq libraries (‘quant’). ~7-fold coverage enrichment is observed at the m1A1322 site together with mutation signatures. d, Overlays of coverage tracks for m1A sites in mRNA and lncRNA. Coverage enrichment of IP libraries is observed for ND5 and MALAT1 sites, however, not for the PRUNE site. e, Lorenz curve analysis of IP libraries as compared to quant libraries without AlkB treatment. Replicate 1 in the m1A-quant-seq is set as the reference for RNA expression level in HEK293T cells; the diagonal line suggests uniform coverage relative to the reference, and more deviated curves suggest more biased coverage distribution. IP libraries show significant biases relative to the quant libraries.
