Abstract
Bacterial RNA sequencing (RNA-seq) is a powerful approach for quantitatively delineating the global transcriptional profiles of microbes in order to gain deeper understanding of their physiology and function. Cost-effective bacterial RNA-seq requires efficient physical removal of ribosomal RNA (rRNA), which otherwise dominates transcriptomic reads. However, current methods to effectively deplete rRNA of diverse non-model bacterial species are lacking. Here, we describe a probe and ribonuclease based strategy for bacterial rRNA removal. We implemented the method using either chemically synthesized oligonucleotides or amplicon-based single-stranded DNA probes and validated the technique on three novel gut microbiota isolates from three distinct phyla. We further showed that different probe sets can be used on closely related species. We provide a detailed methods protocol, probe sets for >5000 common microbes from RefSeq, and an online tool to generate custom probe libraries. This approach lays the groundwork for large-scale and cost-effective bacterial transcriptomics studies.
Highlights
Bacterial RNA sequencing (RNA-seq) provides global and in-depth transcriptional profiling of a given species or community that can yield quantitative and mechanistic insights into microbial function and ecology, and holds exciting potential for routine use in microbiology studies and large-scale screening applications[1,2,3,4,5,6,7]
The 16S and 23S ribosomal RNA are the most abundant RNA molecules in the cell and typically account for more than 90% of the total RNA[8]. These rRNA need to be experimentally removed from total RNA during library preparation steps to ensure cost-effective bacterial RNA-seq with sufficient coverage of coding sequences of the transcriptome
We show that our approach efficiently depletes bacterial rRNA with minimal off-target effects to the transcriptome and demonstrate the use of the method on bacterial species from three different phyla
Summary
Bacterial RNA-seq provides global and in-depth transcriptional profiling of a given species or community that can yield quantitative and mechanistic insights into microbial function and ecology, and holds exciting potential for routine use in microbiology studies and large-scale screening applications[1,2,3,4,5,6,7]. MICROBExpress (Ambion), RiboMinus (Life Technologies) and riboPOOL (siTOOLs Biotech) utilize probes targeting 16S and 23S rRNA to capture rRNA; mRNAONLY (Epicentre) uses a 5 ́-monophosphate-dependent exonuclease to degrade bacterial rRNA molecules; and Ovation Prokaryotic RNA-seq System (NuGEN) uses selective primer sets to avoid targeting rRNA in cDNA synthesis. These kits can be used to deplete bacterial rRNA for single-species RNA-seq and environmental metatranscriptome profiling, and a subset can be used with custom designed probes to target non-model species[19,20,21,22]
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