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Analysis of Endo-siRNAs in Drosophila

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Animal Endo-SiRNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1173))

Abstract

The small RNA silencing system is an important player in the control of gene expression. In particular analysis of the highly complex sequence repertoire of piRNAs and endogenous siRNAs directed against repetitive elements and transposons has been facilitated by the advent of next-generation sequencing tools. We are providing a detailed protocol for the creation of deep sequencing libraries in combination with a chemical modification step (periodate oxidation and β-elimination) that enriches for 3′-terminal modified small RNAs. This step can thus facilitate the detection and—since incorporation of the small RNA into the effector complex precedes the terminal methylation—determine the Argonaute-loading state of certain small RNA species. Both, the oxidation as well as the deep sequencing library preparation are adaptations of popular, previously published protocols. The sequencing libraries described here can be sequenced on all flow cell types (i.e., single-end and paired-end) of the Illumina sequencing systems and are compatible with Illumina index reads. It is therefore also possible to analyze the libraries on the MiSeq system, for which currently only paired-end flow cells are available, and combine several libraries in multiplexed experiments. The chapter concludes with considerations concerning quality control and data analysis.

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Acknowledgements

We would like to thank Romy Böttcher for help with experiments to validate the deep sequencing protocol.

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    Authors and Affiliations

    1. Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, D-81377, Munich, Germany

      Katharina Elmer, Stephanie Helfer, Milijana Mirkovic-Hösle & Klaus Förstemann

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    1. Katharina Elmer
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    2. Stephanie Helfer
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    3. Milijana Mirkovic-Hösle
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    4. Klaus Förstemann
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    Corresponding author

    Correspondence to Klaus Förstemann .

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    Editors and Affiliations

    1. Institute for Cell and Molecular Bioscie, Newcastle University, United Kingdom

      Andreas Werner

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    Elmer, K., Helfer, S., Mirkovic-Hösle, M., Förstemann, K. (2014). Analysis of Endo-siRNAs in Drosophila. In: Werner, A. (eds) Animal Endo-SiRNAs. Methods in Molecular Biology, vol 1173. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0931-5_4

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    • DOI: https://doi.org/10.1007/978-1-4939-0931-5_4

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    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-0930-8

    • Online ISBN: 978-1-4939-0931-5

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