5-Formylcytidine (fC) is one type of post-transcriptional RNA modification, which is known at the wobble position of tRNA in mitochondria and essential for mitochondrial protein synthesis. Here, we show a method to detect fC modifications in RNA and a transcriptome-wide fC mapping technique, named fC-seq. It is developed based on the treatment of pyridine borane, which can reduce fC to 5,6-dihydrouracil, thus inducing C-to-T transition in fC sites during PCR to achieve single-base resolution detection. More than 1000 fC sites were identified after mapping in by fC-seq. Moreover, codon composition demonstrated a preference for fC within wobble sites in mRNA, suggesting the potential role in regulation of translation. These findings expand the scope of the understanding of cytosine modifications in mRNA.
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