Abstract
Next generation sequencing (NGS) is a powerful method for transcriptome analysis. Unlike other gene expression profiling methods, such as microarrays, NGS provides additional information such as splicing variants, sequence polymorphisms, and novel transcripts. For this reason, NGS is well suited for comprehensive profiling of the wide range of extracellular RNAs (exRNAs) in biofluids. ExRNAs are of great interest because of their possible biological role in cell-to-cell communication and for their potential use as biomarkers or for therapeutic purposes. Here, we describe a modified protocol for preparation of small RNA libraries for NGS analysis. This protocol has been optimized for use with low-input exRNA-containing samples, such as plasma or serum, and has modifications designed to reduce the sequence-specific bias typically encountered with commercial small RNA library construction kits.
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References
Baran-Gale J, Kurtz CL, Erdos MR, Sison C, Young A, Fannin EE, Chines PS, Sethupathy P (2015) Addressing bias in small RNA library preparation for sequencing: a new protocol recovers microRNAs that evade capture by current methods. Front Genet 6:352. https://doi.org/10.3389/fgene.2015.00352
Fuchs RT, Sun Z, Zhuang F, Robb GB (2015) Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure. PLoS One 10(5):e0126049. https://doi.org/10.1371/journal.pone.0126049
Jayaprakash AD, Jabado O, Brown BD, Sachidanandam R (2011) Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res 39(21):e141. https://doi.org/10.1093/nar/gkr693
Zhang Z, Lee JE, Riemondy K, Anderson EM, Yi R (2013) High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing. Genome Biol 14(10):R109. https://doi.org/10.1186/gb-2013-14-10-r109
Sorefan K, Pais H, Hall AE, Kozomara A, Griffiths-Jones S, Moulton V, Dalmay T (2012) Reducing ligation bias of small RNAs in libraries for next generation sequencing. Silence 3(1):4. https://doi.org/10.1186/1758-907X-3-4
Viollet S, Fuchs RT, Munafo DB, Zhuang F, Robb GB (2011) T4 RNA ligase 2 truncated active site mutants: improved tools for RNA analysis. BMC Biotechnol 11:72. https://doi.org/10.1186/1472-6750-11-72
Song Y, Liu KJ, Wang TH (2014) Elimination of ligation dependent artifacts in T4 RNA ligase to achieve high efficiency and low bias microRNA capture. PLoS One 9(4):e94619. https://doi.org/10.1371/journal.pone.0094619
Hafner M, Renwick N, Brown M, Mihailovic A, Holoch D, Lin C, Pena JT, Nusbaum JD, Morozov P, Ludwig J, Ojo T, Luo S, Schroth G, Tuschl T (2011) RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA 17(9):1697–1712. https://doi.org/10.1261/rna.2799511
Acknowledgements
This work was supported in part by the NIH Common Fund, Extracellular RNA Communication Consortium (ERCC) 1U01HL126496-01. The authors would like to thank Maria D. Giraldez for helpful suggestions in the preparation of this manuscript.
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Etheridge, A., Wang, K., Baxter, D., Galas, D. (2018). Preparation of Small RNA NGS Libraries from Biofluids. In: Patel, T. (eds) Extracellular RNA. Methods in Molecular Biology, vol 1740. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7652-2_13
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DOI: https://doi.org/10.1007/978-1-4939-7652-2_13
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7651-5
Online ISBN: 978-1-4939-7652-2
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