Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation

Springer Science and Business Media LLC - Tập 25 - Trang 1-19 - 2023
Celine Everaert1,2, Jasper Verwilt1,2, Kimberly Verniers1,2, Niels Vandamme2,3, Alvaro Marcos Rubio1,2, Jo Vandesompele1,2, Pieter Mestdagh1,2
1Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
2Cancer Research Institute Ghent, Ghent University, Ghent, Belgium
3VIB Single Cell Core, Vlaams Instituut voor Biotechnologie, Ghent-Leuven, Belgium

Tóm tắt

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. 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. 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.

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