Abstract
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.
Highlights
Massive parallel sequencing has become the gold standard for transcriptome analyses and has been employed in organisms as diverse as humans[1], Trypanosoma brucei[2] and Escherichia coli[3]
Sequencing total RNA, we found that 28S alpha, 28S beta and 18S are the most abundant ribosomal RNA (rRNA) transcripts in T. brucei, contributing to 75% of the total T. brucei RNA (Supplementary Table 1)
We describe the establishment of a highly specific and efficient approach to deplete rRNA from total RNA extracts that can be adapted to remove any species-specific rRNA or other abundant transcripts that may mask signal from low abundance transcripts
Summary
Massive parallel sequencing has become the gold standard for transcriptome analyses and has been employed in organisms as diverse as humans[1], Trypanosoma brucei[2] and Escherichia coli[3]. One of two strategies is used to remove rRNA from total RNA, enrichment of mature polyadenylated (poly(A)) mRNA or targeted depletion of rRNA The former is based on the use of oligo(dT) primers during reverse transcription of RNA into cDNA. This is the case for partially degraded samples[6], many short and long ncRNAs7, newly transcribed, unprocessed transcripts[8] or RNA from bacteria[9] To study these classes of RNA by RNA-seq, hybridization-based rRNA depletion is typically performed using one of the available kits that follow two approaches. To ensure efficient rRNA depletion and minimal removal of unrelated transcripts, both approaches require a high degree of sequence homology between rRNA transcripts and DNA probes This restricts the use of the available commercial kits to organisms with rRNA sequences matching those of the provided probes. Using a set of only 12 biotinylated DNA oligos, we were able to reduce the levels of the most abundant rRNA transcripts to less than 5% of the total RNA with minimal off-target effects
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