ena-STUDY-INSTITUTE FOR MOLECULAR INFECTION BIOLOGY-08-03-2019-08:04:20:964-293
We implemented an oligo-based rRNA removal approach and analyzed its efficiency using total RNA extracts from wild type Trypanosoma brucei (Lister 427 bloodstream-form MITat 1.2).
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, mature poly-adenylated transcripts are typically enriched or rRNA molecules are depleted. Targeted depletion of rRNA or other highly abundant transcripts is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNA and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, the 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 poly-adenylated transcripts is not an option and commercial kits for rRNA are not available.
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, mature poly-adenylated transcripts are typically enriched or rRNA molecules are depleted. Targeted depletion of rRNA or other highly abundant transcripts is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNA and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, the 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 poly-adenylated transcripts is not an option and commercial kits for rRNA are not available.