Fig. 1: Transcriptome-wide high-confidence m6A profiling with systematic calibration.
a Diagram of the systematic calibration process using a synthetic modification-free RNA library. b Gel image of total RNA, endogenous mRNA, and IVT RNA, showing a close resemblance between endogenous mRNA and IVT mRNA. This experiment was independently repeated once with similar results. c LC–MS/MS results showing that m6A modifications were not detected in IVT RNA. d Transcript levels showing a high correlation between endogenous mRNA and IVT RNA abundances. TPM, transcripts per million. e Metagene plot of read coverage from the 5′ end to the 3′ end of the transcripts, revealing the full-length coverage of IVT RNA. f Metagene profiles showing the distribution of peaks from the WT mRNA and IVT RNA m6A-seq samples. g Motif analysis of peaks identified from the IVT RNA m6A-seq data. P value was calculated using a one-tailed hypergeometric test. h Venn diagram showing the overlap of m6A peaks between the WT mRNA and the corresponding IVT RNA m6A-seq libraries under +P and −P conditions. Approximately 16% and 11% of m6A peaks of the +P and −P WT mRNA libraries, respectively, are false-positive peaks. i Cumulative reads for one representative gene locus of a false-positive peak and a high-confidence (High-conf.) m6A peak, respectively. 5′ and 3′ indicate the direction of the gene. j Metagene profiles showing the distribution of peaks of uncalibrated, calibrated, and false-positive peaks from the WT samples. The calibrated peaks show a sharper distribution than the uncalibrated peaks. Source data are provided as a Source Data file.
