Efficient and specific oligo-based depletion of rRNA

• 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: 38; 38/91; 45; 45/91; 631/1647/514/1949; 631/326/417/1716; 631/337/2019; 64; 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
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-48692-2

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.