Base-resolution analysis of 5-hydroxymethylcytidine by selective oxidation and reverse transcription arrest

While 5-hydroxymethylcytidine in RNA (hm5C) is associated with cellular development and differentiation, its distribution and biological function remain largely unexplored because suitable detection methods are lacking. Here, we report a base-resolution sequencing method for hm5C in RNA by applying peroxotungstate-mediated chemical conversion of hm5C to trihydroxylated thymine (thT). Reverse transcription by SuperScript III terminated at the thT site, probably because of its unnatural nucleobase structure producing truncated cDNA. Consequently, base-resolution analysis of the hm5C sites in RNA was achieved with both Sanger sequencing and Illumina sequencing analysis by comparing sequencing data before and after peroxotungstate treatment.