Advertisement

Small RNA Library Construction for High-Throughput Sequencing

  • Jon McGinn
  • Benjamin Czech
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1093)

Abstract

Since their discovery about 20 years ago, small RNAs have been shown to play a critical role in a myriad of biological processes. The greater availability of high-throughput sequencing has been invaluable to furthering our understanding of small RNAs as regulatory molecules. In particular, these sequencing technologies have been crucial in understanding the role of small RNAs in reproductive tissues, where millions of individual sequences are generated. In this context, high-throughput sequencing provides the requisite level of resolution that other procedures, like northern blotting, would not be able to achieve. Here, we describe a protocol for the preparation of small RNA libraries for sequencing using the Solexa/Illumina technology.

Key words

Endo-siRNA miRNAs piRNAs Small RNA libraries Cloning High-throughput sequencing 

Notes

Acknowledgments

We thank past and current members of the Hannon laboratory that helped improve this protocol, especially Alexei Aravin, Julius Brennecke, and Colin Malone.

References

  1. 1.
    Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843–854PubMedCrossRefGoogle Scholar
  2. 2.
    Wightman B, Ha I, Ruvkun G (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75:855–862PubMedCrossRefGoogle Scholar
  3. 3.
    Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858PubMedCrossRefGoogle Scholar
  4. 4.
    Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862PubMedCrossRefGoogle Scholar
  5. 5.
    Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864PubMedCrossRefGoogle Scholar
  6. 6.
    Aravin A et al (2006) A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442:203–207PubMedGoogle Scholar
  7. 7.
    Girard A, Sachidanandam R, Hannon GJ, Carmell MA (2006) A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442:199–202PubMedGoogle Scholar
  8. 8.
    Grivna ST, Beyret E, Wang Z, Lin H (2006) A novel class of small RNAs in mouse spermatogenic cells. Genes Dev 20:1709–1714PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Lau NC et al (2006) Characterization of the piRNA complex from rat testes. Science 313:363–367PubMedCrossRefGoogle Scholar
  10. 10.
    Ruby JG et al (2006) Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127:1193–1207PubMedCrossRefGoogle Scholar
  11. 11.
    Brennecke J et al (2007) Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128:1089–1103PubMedCrossRefGoogle Scholar
  12. 12.
    Gunawardane LS et al (2007) A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila. Science 315:1587–1590PubMedCrossRefGoogle Scholar
  13. 13.
    Houwing S et al (2007) A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish. Cell 129:69–82PubMedCrossRefGoogle Scholar
  14. 14.
    Pfeffer S, Lagos-Quintana M, Tuschl T (2005) Cloning of small RNA molecules. Curr Protoc Mol Biol Chapter 26, Unit 26.4, edited by Frederick M. Ausubel … [et al.]Google Scholar
  15. 15.
    Hafner M et al (2008) Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44:3–12PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Zhuang F, Fuchs RT, Robb GB (2012) Small RNA expression profiling by high-throughput sequencing: implications of enzymatic manipulation. J Nucleic Acids 2012:360358Google Scholar
  17. 17.
    Silber R, Malathi VG, Hurwitz J (1972) Purification and properties of bacteriophage T4-induced RNA ligase. Proc Natl Acad Sci U S A 69:3009–3013PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Jon McGinn
    • 1
  • Benjamin Czech
    • 1
  1. 1.Cold Spring Harbor LaboratoryCold Spring HarborUSA

Personalised recommendations