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

Abstract

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.