Species independent rRNA removal

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

rna-seq

(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.

Kraus AJ, Brink BG, Siegel TN. (2019) Efficient and specific oligo-based depletion of rRNA. Sci Rep 9(1):12281. [article]

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