Abstract
Small RNAs (smRNAs) play an essential role in virtually every aspect of growth and development, by regulating gene expression at the post-transcriptional and/or transcriptional level. New high-throughput sequencing technology allows for a comprehensive coverage of smRNAs in any given biological sample, and has been widely used for profiling smRNA populations in various developmental stages, tissue and cell types, or normal and disease states. In this article, we describe the method used in our laboratory to construct smRNA cDNA libraries for high-throughput sequencing.
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References
Carthew, R. W., and Sontheimer, E. J. (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136, 642–655.
Voinnet, O. (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136, 669–687.
Kim, V. N., Han, J., and Siomi, M. C. (2009) Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell. Biol. 10, 126–139.
Okamura, K., Chung, W. J., and Lai, E. C. (2008) The long and short of inverted repeat genes in animals: microRNAs, mirtrons and hairpin RNAs. Cell Cycle 7, 2840–2845.
Ramachandran, V., and Chen, X. (2008) Small RNA metabolism in Arabidopsis. Trends Plant Sci. 13, 368–374.
Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001) Identification of novel genes coding for small expressed RNAs. Science 294, 853–858.
Lau, N. C., Lim, L. P., Weinstein, E. G., and Bartel, D. P. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862.
Lee, R. C., and Ambros, V. (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862–864.
Fahlgren, N., Sullivan, C. M., Kasschau, K. D., Chapman, E. J., Cumbie, J. S., Montgomery, T. A., Gilbert, S. D., Dasenko, M., Backman, T. W., Givan, S. A., and Carrington, J. C. (2009) Computational and analytical framework for small RNA profiling by high-throughput sequencing. RNA 15, 992–1002.
Sharma, C. M., and Vogel, J. (2009) Experimental approaches for the discovery and characterization of regulatory small RNA. Curr. Opin. Microbiol. 12, 536–546.
Lu, C., Tej, S. S., Luo, S., Haudenschild, C. D., Meyers, B. C., and Green, P. J. (2005) Elucidation of the small RNA component of the transcriptome. Science 309, 1567–1569.
Lister, R., Gregory, B. D., and Ecker, J. R. (2009) Next is now: new technologies for sequencing of genomes, transcriptomes, and beyond. Curr. Opin. Plant Biol. 12, 107–118.
Shendure, J., Mitra, R. D., Varma, C., and Church, G. M. (2004) Advanced sequencing technologies: methods and goals. Nat. Rev. Genet. 5, 335–344.
Girard, A., Sachidanandam, R., Hannon, G. J., and Carmell, M. A. (2006) A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442, 199–202.
Qi, Y., He, X., Wang, X. J., Kohany, O., Jurka, J., and Hannon, G. J. (2006) Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443, 1008–1012.
Katiyar-Agarwal, S., Gao, S., Vivian-Smith, A., and Jin, H. (2007) A novel class of bacteria-induced small RNAs in Arabidopsis. Genes Dev. 21, 3123–3134.
Lee, Y. S., Shibata, Y., Malhotra, A., and Dutta, A. (2009) A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). Genes Dev. 23, 2639–2649.
Addo-Quaye, C., Eshoo, T. W., Bartel, D. P., and Axtell, M. J. (2008) Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr. Biol. 18, 758–762.
German, M. A., Pillay, M., Jeong, D. H., Hetawal, A., Luo, S., Janardhanan, P., Kannan, V., Rymarquis, L. A., Nobuta, K., German, R., De Paoli, E., Lu, C., Schroth, G., Meyers, B. C., and Green, P. J. (2008) Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends. Nat. Biotechnol. 26, 941–946.
Accerbi, M., Schmidt, S. A., De Paoli, E., Park, S., Jeong, D. H., and Green, P. J. (2010) Methods for isolation of total RNA to recover miRNAs and other small RNAs from diverse species. Methods Mol. Biol. 592, 31–50.
Ho, C. K., and Shuman, S. (2002) Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains. Proc. Natl. Acad. Sci. USA 99, 12709–12714.
Ho, C. K., Wang, L. K., Lima, C. D., and Shuman, S. (2004) Structure and mechanism of RNA ligase. Structure 12, 327–339.
Aravin, A., and Tuschl, T. (2005) Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett. 579, 5830–5840.
Hafner, M., Landgraf, P., Ludwig, J., Rice, A., Ojo, T., Lin, C., Holoch, D., Lim, C., and Tuschl, T. (2008) Identification of microÂRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44, 3–12.
Acknowledgments
The authors would like to thank Dr. Pamela Green for encouragement and mentoring on the smRNA study. The work was done in her laboratory. The research was supported by USDA grant #2007-1-0199 to P.J. Green.
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Lu, C., Shedge, V. (2011). Construction of Small RNA cDNA Libraries for High-Throughput Sequencing. In: Lu, C., Browse, J., Wallis, J. (eds) cDNA Libraries. Methods in Molecular Biology, vol 729. Humana Press. https://doi.org/10.1007/978-1-61779-065-2_9
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DOI: https://doi.org/10.1007/978-1-61779-065-2_9
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