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

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RNA Modifications

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2298))

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.

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Acknowledgments

This work was supported by the National Center for Scientific Research (CNRS), the French Alternative Energies and Atomic Energy Commission (CEA), and Paris-Sud University. The members of the I2BC Next-Generation Sequencing service are acknowledged for critical reading of the manuscript and helpful suggestions.

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Authors and Affiliations

  1. Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud, Université Paris-Saclay, Gif sur Yvette Cedex, France

    Erwin L. van Dijk & Claude Thermes

Authors
  1. Erwin L. van Dijk
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  2. Claude Thermes
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Corresponding author

Correspondence to Erwin L. van Dijk .

Editor information

Editors and Affiliations

  1. Department of Urology, University of California, San Francisco, San Francisco, CA, USA

    Mary McMahon

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van Dijk, E.L., Thermes, C. (2021). A Small RNA-Seq Protocol with Less Bias and Improved Capture of 2′-O-Methyl RNAs. In: McMahon, M. (eds) RNA Modifications. Methods in Molecular Biology, vol 2298. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1374-0_10

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  • DOI: https://doi.org/10.1007/978-1-0716-1374-0_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1373-3

  • Online ISBN: 978-1-0716-1374-0

  • eBook Packages: Springer Protocols

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