Base-resolution m 5 C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach

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