Protocol for single-base quantification of RNA m5C by pyrosequencing

5-methylcytosine (m5C) is a common RNA modification found in both coding and non-coding RNAs. Here, we present a protocol for RNA-m5C-pyroseq for quantitative, single-nucleotide resolution analysis of RNA methylation. We describe steps for designing primers for the target region of RNA, performing bisulfite conversion of RNA, and using pyrosequencing to measure RNA methylation in cytosine. We then detail procedures for all the steps, including RNA isolation, bisulfite conversion, cDNA synthesis, PCR, gel electrophoresis, and pyrosequencing.