Author:
Everaert Celine,Verwilt Jasper,Verniers Kimberly,Vandamme Niels,Marcos Rubio Alvaro,Vandesompele Jo,Mestdagh Pieter
Abstract
Abstract
Background
RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species.
Results
We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3′ end sequencing and long-read sequencing, and MALAT1 in single-cell 3′ end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity.
Conclusion
Our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.
Publisher
Springer Science and Business Media LLC
Subject
General Biochemistry, Genetics and Molecular Biology
Reference70 articles.
1. O’Neil D, Glowatz H, Schlumpberger M. Ribosomal RNA Depletion for Efficient Use of RNA-Seq Capacity. Curr Protoc Mol Biol. 2013:4.19.1–8 Available from: http://doi.wiley.com/10.1002/0471142727.mb0419s103. Cited 2020 Apr 22. Hoboken, NJ, USA: John Wiley & Sons, Inc.
2. Hendrick JP, Wolin SL, Rinke J, Lerner MR, Steitz JA. Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells. Mol Cell Biol. American Society for Microbiology. 1981;1:1138–49.
3. Rutjes SA, Van Der Heijden A, Utz PJ, Van Venrooij WJ, Pruijn GJM. Rapid nucleolytic degradation of the small cytoplasmic Y RNAs during apoptosis. J Biol Chem. American Society for Biochemistry and Molecular Biology. 1999;274:24799–807.
4. Nicolas FE, Hall AE, Csorba T, Turnbull C, Dalmay T. Biogenesis of Y RNA-derived small RNAs is independent of the microRNA pathway. FEBS Lett. 2012;586:1226–30 Available from: http://doi.wiley.com/10.1016/j.febslet.2012.03.026. Cited 2020 Mar 30. John Wiley & Sons, Ltd.
5. Meiri E, Levy A, Benjamin H, Ben-David M, Cohen L, Dov A, et al. Discovery of microRNAs and other small RNAs in solid tumors. Nucleic Acids Res. 2010;38:6234–46 Available from: http://www.agilent.com. Cited 2020 Apr 24.
Cited by
1 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献