Abstract
The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ∼75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest.IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a "do-it-yourself" kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.
Highlights
The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria
Our results indicate that the kit we developed enables the facile depletion of rRNA from total RNA samples such that ϳ70 to 80% of reads in RNA-seq map to mRNAs
To efficiently and inexpensively deplete rRNA from total RNA from multiple organisms, we developed an algorithm to design DNA oligonucleotides capable of hybridizing to rRNA from multiple species simultaneously
Summary
The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. To enrich for mRNA in RNA-seq samples, a general strategy involves the depletion of rRNAs by subtractive hybridization [8,9,10] This approach was at the heart of commercially available kits such as Ribo-Zero from Illumina, leading to RNA-seq data in which ϳ80 to 90% of the reads map to mRNAs. Despite the popularity and efficacy of Ribo-Zero, this kit was abruptly discontinued by the manufacturer for an extended period of time, which disrupted many RNA-seq users and pipelines that depend on it. Our results indicate that the kit we developed enables the facile depletion of rRNA from total RNA samples such that ϳ70 to 80% of reads in RNA-seq map to mRNAs. We further demonstrate that our kit produces RNA-seq data showing high correspondence to that produced using the Ribo-Zero kit. We anticipate that this rRNA-depletion strategy will benefit the entire bacterial community by enabling low-cost transcriptomics with a similar workflow as the Ribo-Zero kit
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