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Analysis of Endo-siRNAs in Drosophila

  • Katharina Elmer
  • Stephanie Helfer
  • Milijana Mirkovic-Hösle
  • Klaus FörstemannEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Keywords

Deep sequencing Next-generation sequencing Illumina TrueSeq Index MiSeq system miRNA Endo-siRNA esiRNA Endogenous siRNA 

Notes

Acknowledgements

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

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Katharina Elmer
    • 1
  • Stephanie Helfer
    • 1
  • Milijana Mirkovic-Hösle
    • 1
  • Klaus Förstemann
    • 1
    Email author
  1. 1.Gene Center and Department of BiochemistryLudwig-Maximilians-Universität MünchenMunichGermany

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