Base-resolution mC profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach.

5-methylcytosine (mC) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive mC sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report mC-TAC-seq, a bisulfite-free approach combining TET-assisted mC-to-fC oxidation with selective chemical labeling, therefore enabling direct base-resolution mC detection through pre-enrichment and C-to-T transitions at mC sites. With mC-TAC-seq, we comprehensively profiled the mC methylomes in human and mouse cells, identifying a substantially larger number of confident mC sites. Through perturbing potential mC methyltransferases, we deciphered the responsible enzymes for most mC sites, including the characterization of NSUN5's involvement in mRNA mC deposition. Additionally, we characterized mC dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in mC-TAC-seq allow mC detection in chromatin-associated RNAs. The accurate and robust mC-TAC-seq will advance research into mC methylation functional investigation.