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Target-enrichment sequencing for detailed characterization of small RNAs

Nature Protocols volume 13, pages 768786 (2018) | Download Citation

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Abstract

Identification of important, functional small RNA (sRNA) species is currently hampered by the lack of reliable and sensitive methods to isolate and characterize them. We have developed a method, termed target-enrichment of sRNAs (TEsR), that enables targeted sequencing of rare sRNAs and diverse precursor and mature forms of sRNAs not detectable by current standard sRNA sequencing methods. It is based on the amplification of full-length sRNA molecules, production of biotinylated RNA probes, hybridization to one or multiple targeted RNAs, removal of nontargeted sRNAs and sequencing. By this approach, target sRNAs can be enriched by a factor of 500–30,000 while maintaining strand specificity. TEsR enriches for sRNAs irrespective of length or different molecular features, such as the presence or absence of a 5′ cap or of secondary structures or abundance levels. Moreover, TEsR allows the detection of the complete sequence (including sequence variants, and 5′ and 3′ ends) of precursors, as well as intermediate and mature forms, in a quantitative manner. A well-trained molecular biologist can complete the TEsR procedure, from RNA extraction to sequencing library preparation, within 4–6 d.

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Change history

  • 23 April 2018

    In the version of this article initially published, the left panel of Figure 4a was erroneously duplicated and used for both the left and right panels. However, these panels should show different graphs. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank all P.C. and F.d.d.F. laboratory members, especially G. Pascarella, K. Hashimoto and A. Bonetti, for insightful discussions. F.d.d.F.'s laboratory is supported by Associazione Italiana per la Ricerca sul Cancro (application 12971), AriSLA (project 'DDRNA and ALS'), Associazione Italiana per la Ricerca sul Cancro (AIRC; application 12971), Worldwide Cancer Research (Association for International Cancer Research (AICR), Research Infrastructure Fund (RIF) 14-1331), Research EPIGEN, Fondazione Cariplo (grants 2014-1215 and 2014-0812), Progetti di Ricerca di Interesse Nazionale (PRIN) 2010–2011, Fondazione Telethon (GGP12059), the Human Frontier Science Program (contract RGP 0014/2012) and a European Research Council advanced grant (322726). P.C.'s lab is supported by a Research Grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to the RIKEN Center for Life Science Technologies. J.A. is supported by Marie Curie Initial Training Networks (FP7 PEOPLE 2012 ITN (CodeAge project no: 316354)). F.R. is supported by Fondazione Italiana per la Ricerca sul Cancro (FIRC, application number 12476).

Author information

Author notes

    • Quan Nguyen

    Present address: Genomics of Development and Disease Division, Institute for Molecular Bioscience, Queensland Bioscience Precinct, St Lucia, Queensland, Australia.

    • Quan Nguyen
    •  & Julio Aguado

    These authors contributed equally to this work.

Affiliations

  1. Division of Genomic Technologies, RIKEN Yokohama Campus, RIKEN Center for Life Science Technologies, Tsurumi-Ku, Yokohama, Japan.

    • Quan Nguyen
    • , Ana Maria Suzuki
    •  & Piero Carninci
  2. IFOM Foundation—FIRC Institute of Molecular Oncology Foundation, Milan, Italy.

    • Julio Aguado
    • , Fabio Iannelli
    • , Francesca Rossiello
    •  & Fabrizio d'Adda di Fagagna
  3. Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), Pavia, Italy.

    • Fabrizio d'Adda di Fagagna

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Contributions

P.C., F.d.d.F. and Q.N. conceived the project. Q.N., J.A. and F.R. developed the protocol. Q.N., J.A. and A.M.S. performed the biochemical experiments. Q.N. and F.I. developed the bioinformatics analysis framework and analyzed the data. Q.N., J.A., F.R., F.d.d.F. and P.C. discussed and interpreted the results. All authors contributed to and approved the writing of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Fabrizio d'Adda di Fagagna or Piero Carninci.

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    Supplementary Figures 1–8 and Supplementary Tables 1 and 2.

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DOI

https://doi.org/10.1038/nprot.2018.001

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