Efficient and specific oligo-based depletion of rRNA

In most organisms, ribosomal RNA (rRNA) contributes to >85% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA...

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Bibliographic Details
Published in:Scientific reports 2019-08, Vol.9 (1), p.12281-8, Article 12281
Main Authors: Kraus, Amelie J., Brink, Benedikt G., Siegel, T. Nicolai
Format: Article
Language:English
Subjects:
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DNA probes
Homology
Humanities and Social Sciences
Hybridization
multidisciplinary
Nucleotide sequence
Oligonucleotides
Oligonucleotides - chemistry
Oligonucleotides - genetics
Organisms
Poly(A)
Polyadenylation
RNA probes
RNA, Protozoan - chemistry
RNA, Protozoan - genetics
RNA, Ribosomal - chemistry
RNA, Ribosomal - genetics
rRNA
Science
Science (multidisciplinary)
Sequence Analysis, RNA
Streptavidin
Trypanosoma cruzi - genetics
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Summary:In most organisms, ribosomal RNA (rRNA) contributes to >85% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences. In this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5% with minimal off-target effects. By adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-019-48692-2