Posttranscriptional RNA modifications have recently emerged as essential posttranscriptional regulators of gene expression. Here we present two methods for single nucleotide resolution detection of 5-formylcytosine (f5C) in RNA. The first relies on chemical protection of f5C against bisulfite treatment, the second method is based on chemical reduction of f5C to hm5C. In combination with regular bisulfite treatment of RNA, the methods allow for precise mapping of f5C. The protocol is used for f5C detection in mtDNA-encoded RNA, however, it can be straightforwardly applied for transcriptome-wide analyses.
Keywords: 5-formylcytosine; Next-generation sequencing; RNA modification; RedBS RNA-Seq; epitranscriptome; fCAB RNA-Seq.