A Small RNA-Seq Protocol with Less Bias and Improved Capture of 2'-O-Methyl RNAs

Erwin L van Dijk; Claude Thermes

doi:10.1007/978-1-0716-1374-0_10

A Small RNA-Seq Protocol with Less Bias and Improved Capture of 2'-O-Methyl RNAs

Methods Mol Biol. 2021:2298:153-167. doi: 10.1007/978-1-0716-1374-0_10.

Authors

Erwin L van Dijk¹, Claude Thermes²

Affiliations

¹ Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud, Université Paris-Saclay, Gif sur Yvette Cedex, France. erwin.vandijk@i2bc.paris-saclay.fr.
² Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud, Université Paris-Saclay, Gif sur Yvette Cedex, France.

PMID: 34085244
DOI: 10.1007/978-1-0716-1374-0_10

Abstract

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNAs such as plant microRNAs (miRNAs) carry a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This modification adds another level of difficulty as it inhibits 3' adapter ligation. We previously demonstrated that modified versions of the "TruSeq (TS)" protocol have less bias and an improved detection of 2'-OMe RNAs. Here we describe in detail protocol "TS5," which showed the best overall performance. We also provide guidelines for bioinformatics analysis of the sequencing data.

Keywords: 2′-O-methyl (2′-OMe) RNA; Bias; Library preparation; NGS; Next-generation sequencing; Plant miRNA; Plant microRNA; Small RNA; Small RNA-seq.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Computational Biology / methods
Gene Library
High-Throughput Nucleotide Sequencing / methods*
MicroRNAs / genetics
RNA, Small Untranslated / genetics*
RNA-Seq / methods*
Sequence Analysis, RNA / methods*

Substances

MicroRNAs
RNA, Small Untranslated