Detection of 5-Methylcytosine in Specific Poly(A) RNAs by Bisulfite Sequencing

RNA bisulfite sequencing (RNA-BS-seq) represents a method for the detection of methylated cytosines in RNA. Developed originally for the analysis of DNA methylation, a modified version of this method can be used for the analysis of methylated cytosine in RNA. Treatment of nucleic acids with HSO₃-ions under acidic conditions results in deamination of cytosine (C) to uracil, while 5-methylcytosine (m5C) or 5-hydroxymethylcytosine (hm5C) exhibit low reactivity in this reaction and remain unchanged. Subsequent PCR amplification and sequencing of specific targets allows for the assessment of the methylation status of single Cs in their native sequence context at nucleotide resolution. Here, we describe the application of this method for the analysis of cytosine methylation in low abundance poly(A)RNA using a combination of commercially available kits and standard lab methods to ensure reproducible results. Furthermore, useful information on optimizing the method, suitable controls for almost all steps, and general troubleshooting guides are provided.