Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach

5-methylcytosine (m⁵C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m⁵C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m⁵C-TAC-seq, a bisulfite-free approach combining TET-assisted m⁵C-to-f⁵C oxidation with selective chemical labeling, therefore enabling direct base-resolution m⁵C detection through pre-enrichment and C-to-T transitions at m⁵C sites. With m⁵C-TAC-seq, we comprehensively profiled the m⁵C methylomes in human and mouse cells, identifying a substantially larger number of confident m⁵C sites. Through perturbing potential m⁵C methyltransferases, we deciphered the responsible enzymes for most m⁵C sites, including the characterization of NSUN5’s involvement in mRNA m⁵C deposition. Additionally, we characterized m⁵C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m⁵C-TAC-seq allow m⁵C detection in chromatin-associated RNAs. The accurate and robust m⁵C-TAC-seq will advance research into m⁵C methylation functional investigation.