Extended Data Fig. 4 ∣. Characterization of specificity and background of picoMeRIP.

a, Top panel, schematic illustration of experimental setup for titration of two control RNAs, m⁶A-modified GLuc, and unmodified CLuc, for picoMeRIP and qPCR assessment. Bottom panel, quantified immunoprecipitation levels of m⁶A-modified GLuc, and unmodified CLuc, after picoMeRIP. picoMeRIP signal was normalized to 100% GLuc. Data are presented as mean ± SD values, with n = 2 independent experiments for each experimental condition. Created with BioRender.com. b, Experimentally observed m⁶A levels of m⁶A-modified control RNA as compared to the expected ones based on the titration experiment described in panel a. Data are presented as mean ± SD values, with n = 2 independent experiments for each experimental condition. R is the correlation coefficient in regression analysis. c, qPCR assessment of picoMeRIP m⁶A signal over background with two control RNAs used as spike-ins, m⁶A-modified GLuc, and unmodified CLuc, and for the previously validated m⁶A positive (Pdzd8) and negative (Rdh10) liver transcripts. Data are presented as mean ± SD values, with n = 3 independent experiments for each experimental condition. d, Western blot of METTL3 and GAPDH for WT and Mettl3 deficient (“knock-out” (KO)) mES cell lines. GAPDH was used as loading control. Two independent western blot experiments were performed with similar results. e, Schematic illustration of experimental setup to compare number of m⁶A peaks and peak signal strength of picoMeRIP-seq from WT and Mettl3 deficient (KO) mES cells, including spiked-in control RNAs. Mouse liver mRNA and mES cell mRNA were added in a 1:1 ratio. Two control RNAs were used as spike-ins, m⁶A-modified GLuc, and unmodified CLuc, and added at a 1:1 ratio. Created with BioRender.com. f, Number of picoMeRIP-seq m⁶A peaks uniquely identified in WT and Mettl3 deficient (KO) mES cells, and co-identified in mouse liver and WT mES cells. g, Box plots of m⁶A signal for comparison of picoMeRIP-seq peaks uniquely identified in WT and Mettl3 deficient (KO) mES cells. Middle vertical line (bold) depicts the median, and the 25^th percentile to 75^th percentile is shown. Whiskers extend to the most extreme data point within 1.5 interquartile range of the quartiles. The p value was calculated using a Wilcoxon rank sum test (two-sided). The corresponding m⁶A peak numbers for each of three groups are shown in panel f. h, Assessment of false discovery rate of picoMeRIP-seq by spike-in m⁶A-modified GLuc, and unmodified Cluc control RNAs.