Base-resolution analysis of 5-hydroxymethylcytidine by selective oxidation and reverse transcription arrest. Issue 29 (9th July 2021)

• Record Type: Journal Article
• Authors: Koyama, Kenta; Hayashi, Gosuke; Ueda, Hiroki; Ota, Satoshi; Nagae, Genta; Aburatani, Hiroyuki; Okamoto, Akimitsu
• Is Part Of: Organic & biomolecular chemistry. Volume 19:Issue 29(2021)
• Page Start: 6478
• Page End: 6486
• Publication Date: 2021-07-09
• Subjects: Chemistry, Organic -- Periodicals; Bioorganic chemistry -- Periodicals; Chemistry, Physical organic -- Periodicals; 547
• DOI: 10.1039/d1ob00995h
• Languages: English
• ISSNs: 1477-0520

Abstract

Abstract : A base-resolution analysis of RNA hm5 C "WT-Seq" has been developed using the peroxotungstate-mediated oxidation and subsequent truncated cDNA production. Abstract : While 5-hydroxymethylcytidine in RNA ( hm5 C) 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 hm5 C in RNA by applying peroxotungstate-mediated chemical conversion of hm5 C to trihydroxylated thymine ( th T). Reverse transcription by SuperScript III terminated at the th T site, probably because of its unnatural nucleobase structure producing truncated cDNA. Consequently, base-resolution analysis of the hm5 C sites in RNA was achieved with both Sanger sequencing and Illumina sequencing analysis by comparing sequencing data before and after peroxotungstate treatment.