Optimization of NaIO4 Oxidation and Deep-sequencing Methodology for Detection of 3' End Methylation of Small RNAs in low-input RNA Samples

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 addition when we stop 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. Overall design: We optimized the oxidation condition, and omitted the glycerol adding when we stop 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