Table 1 Approaches for the mapping of m5C in RNA

Bisulfite sequencing

Unmethylated Cs are deaminated to Us in the presence of sodium bisulfite, while methylated Cs remain unchanged

Unbiased transcriptome-wide mapping at single nucleotide resolution and with high specificity

Unable to detect m⁵C sites in low-abundance RNAs, and fail to distinguish m⁵C from other types of cytosine modifications

Single nucleotide resolution

RIP-seq

RNA immunoprecipitation

Specific mapping of m⁵C sites in low abundance RNAs

Not at single nucleotide resolution

100–150 nt

Aza-IP-seq

Protein immunoprecipitation

Investigate specific catalytic sites of RCTMs

Missing of unstably converted m⁵C sites, random incorporation of cytidine analogue to DNAs, and toxicity to cells

Enzyme-specific nucleotide resolution

miCLIP-seq

Protein immunoprecipitation

Investigate specific catalytic sites of RCTMs

Time-consuming and costly

Enzyme-specific nucleotide resolution