An Improved NaIO4 Oxidation and Deep-Sequencing Method for Detecting 3′ Terminal Methylation of Small RNAs in Low-Input RNA Samples

XUE Wanqiang^1,2, HOU Li², LI Ronghong², WU Ligang^2*

(¹School of Life Science, Shanghai University, Shanghai 200444, China; ²State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China)

Abstract:

The 2′-O-methylation is present at the 3′ end of various small RNAs and has important biological functions. At present, NaIO4 oxidation treatment combined with deep sequencing is one of the most powerful method to study the 2′-O-methylation of small RNAs. However, currently used protocol requires 3 μg of total RNA and is not suitable for studying 3′ terminal 2′-O-methylation of small RNAs in limited number of cells. In this study, we optimized the oxidation condition and omitted the glycerol when stops the oxidation reaction. With these improvements, we are able to identify the small RNAs bearing 3′ terminal 2′-O-methylation using as little as 10 ng total RNA from mouse testis, providing a robust protocol suitable for investigating 3′ terminal 2′-O-methylation of small RNAs in a small number of cells.

CSTR: 32200.14.cjcb.2019.09.0012