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.
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References
Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843–854
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 75:855–862
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858
Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862
Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864
Aravin A et al (2006) A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442:203–207
Girard A, Sachidanandam R, Hannon GJ, Carmell MA (2006) A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442:199–202
Grivna ST, Beyret E, Wang Z, Lin H (2006) A novel class of small RNAs in mouse spermatogenic cells. Genes Dev 20:1709–1714
Lau NC et al (2006) Characterization of the piRNA complex from rat testes. Science 313:363–367
Ruby JG et al (2006) Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127:1193–1207
Brennecke J et al (2007) Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128:1089–1103
Gunawardane LS et al (2007) A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila. Science 315:1587–1590
Houwing S et al (2007) A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish. Cell 129:69–82
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.]
Hafner M et al (2008) Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44:3–12
Zhuang F, Fuchs RT, Robb GB (2012) Small RNA expression profiling by high-throughput sequencing: implications of enzymatic manipulation. J Nucleic Acids 2012:360358
Silber R, Malathi VG, Hurwitz J (1972) Purification and properties of bacteriophage T4-induced RNA ligase. Proc Natl Acad Sci U S A 69:3009–3013
Acknowledgments
We thank past and current members of the Hannon laboratory that helped improve this protocol, especially Alexei Aravin, Julius Brennecke, and Colin Malone.
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McGinn, J., Czech, B. (2014). Small RNA Library Construction for High-Throughput Sequencing. In: Siomi, M. (eds) PIWI-Interacting RNAs. Methods in Molecular Biology, vol 1093. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-694-8_16
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DOI: https://doi.org/10.1007/978-1-62703-694-8_16
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-693-1
Online ISBN: 978-1-62703-694-8
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