| Autor: |
Kraus AJ; Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-Universität München, 80802, Munich, Germany.; Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität München, 82152, Planegg-Martinsried, Germany.; Research Center for Infectious Diseases, University of Würzburg, 97080, Würzburg, Germany., Brink BG; Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-Universität München, 80802, Munich, Germany.; Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität München, 82152, Planegg-Martinsried, Germany., Siegel TN; Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-Universität München, 80802, Munich, Germany. n.siegel@lmu.de.; Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität München, 82152, Planegg-Martinsried, Germany. n.siegel@lmu.de.; Research Center for Infectious Diseases, University of Würzburg, 97080, Würzburg, Germany. n.siegel@lmu.de. |
| Abstrakt: |
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. |
