Detection of 5-formylcytosine in Mitochondrial Transcriptome

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.