Total RNA extraction from the cell lines was accomplished using the RNA isolater total RNA extraction reagent (Vazyme, R401-01). Prior to size selection, RNA underwent DNase I treatment at 37 ℃ for 15 min and subsequently was purified using RNA isolater extraction and ethanol precipitation (Vazyme, EN401-01). The small RNA fraction, characterized by a size of less than 200nt, was enriched from the total RNA utilizing the RNA Clean & Concentrator-25 Kit (Zymo, R1018). For mRNA purification, two successive rounds of poly(A)+ selection were performed employing oligo(dT)25 dynabeads (Life Technologies, 61005). Additionally, chromatin-associated RNA was isolated following subcellular fractionation assay, DNase I treatment, and rRNA depletion (Vazyme, N406-01). Modification-free control RNAs were prepared from HeLa or mESC mRNA, adhering to a previously published protocol (PMID: 34594034, 36593412). The RNA samples were initially dephosphorylated using T4 polynucleotide kinase (Vazyme, N102-01) at 37℃ for 1 hour, followed by ethanol precipitation. Subsequently, they were ligated to the 3' RNA linker using T4 RNA ligase 2, truncated at 25°C for 2 hours, followed by an overnight incubation at 16°C (Beyotime, R0635L). To remove excess adaptors, the samples underwent treatment with 5´ Deadenylase (NEB, M0331S), RecJf (NEB, M0264S), and Proteinase K (NEB, P8102). RNA samples intended for group comparisons were pooled and then purified through Tris-phenol extraction followed by ethanol precipitation. The barcoded and pooled samples were labeled by 5-(2-azidoethyl)-1,3-indandione (AI) and subsequently subjected to a click reaction. The m5C-modified RNA was subjected to RNA pull-down to be enriched, followed by reverse transcription using the HiScript III RT SuperMix (Vazyme, R323-01). The excess reverse transcription primer was digested with exonuclease I (NEB, M0293S). The cDNA was purified by silane beads and then performed to 5′ adapter ligation on beads with T4 RNA ligase 1, high concentration (NEB, M0437M). cDNA was purified using silane beads (Invitrogen, 37002D), and subsequently, the purified cDNA was amplified using the KAPA HiFi HotStart ReadyMix (Roche, KK2602). Finally, PCR products were purified using 1.8X VAHTS DNA Clean Beads (Vazyme, N411-02) and 7% TBE gel.