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 researchers from Ludwig-Maximilians-Universität München describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. This 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, this approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, this 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.

Depletion of rRNA transcripts

(a) Outline of depletion strategy. (b,c) Agarose gels revealing presence or absence of the three large trypanosomal rRNA transcripts following different depletion conditions. For each condition 2 µg of total RNA was used. (d) The samples shown in (c) were analyzed by RNA-seq. Shown are the percentages of rRNA (28S, 5.8S, 18S and 5S) relative to total RNA.