Protocol for detecting m5C RNA modification at single-base resolution using m5C-TAC-seq

Here we report m5C-TAC-seq, a base-resolution sequencing method to directly detect m5C sites without affecting unmodified C. In m5C-seq, we combined TET-mediated oxidation of RNA m5C to f5C with the selective chemical labeling reaction of f5C, enabling both pre-enrichment of m5C-containing RNA and a m5C-to-T transition in reverse transcription. m5C-seq identifies 2,500 sites in the transcriptome of HeLa and 768 sites in the transcriptome-wide of HEK293T. In addition, taking advantage of barcoding and pooling strategy, m5C-seq detected differential m5C sites upon specific methyltransferases depletion in mESCs and dynamically regulated m5C sites under cell fate transition. Moreover, we also detected 215 sites in chromatin-associated RNAs, demonstrating that portion of m5C sites can be co-transcriptionally catalyzed and the existence of m5C methylations in repeat RNAs. To explore m5C distribution in mRNA,we build a TET2-assisted m5C single base solution detect method *** Please note that multiplexed raw files are designed to a protocol to repeat the data and, therefore, have been uploaded to SRA with special exception.