Fig. 1: Systematic mapping of m¹A sites in small RNA space.

a Chemical structure of m¹A (N¹-methyladenosine). b Overall m¹A mapping strategy for small RNAs by combining m¹A RIP and m¹A-induced mismatch analysis. Synthetic m¹A RNA is included as a positive control. c TGIRT identified as the optimal reverse transcriptase for m¹A-induced mismatch analysis. Briefly, synthetic m¹A-containing RNA at different m¹A stoichiometry was sequenced by three different reverse transcriptases (RTs): TGIRT (thermostable group II intron reverse transcriptase), PSII (ProtoScriptII, retrovirus RT) and RT-1306 (engineered HIV RT). For each RT, mismatch rate was calculated across all the reads that map to the synthetic RNA sequence (allowing 1 mismatch) as represented by the sequence logo. The mismatch rate at the known m¹A site is then plotted against known m¹A stoichiometry (R² derived from linear fitting forced to cross intercept at zero). d ProtoScriptII leads to under-representation of m¹A-containing RNAs. Cloning frequency is normalized to spike-ins. e m¹A RIP successfully enriches synthetic m¹A-containing RNA compared to input, when spiked in with total short RNAs. TGIRT captures enrichment better than ProtoScriptII. Data are based on four independent RIP experiments (two HEK293T and two U251). Boxplot center represents median, bounds represent 25 and 75%, and whiskers show the minimum or maximum no further than 1.5 * interquartile range from the bound. See also Supplementary Fig. 1 and Supplementary Table 1. Source data are provided as a Source Data file.