Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion
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METHODOLOGY ARTICLE
Open Access
Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion Chatarin Wangsanuwat1,2†, Kellie A. Heom1,2†, Estella Liu1, Michelle A. O’Malley1,2 and Siddharth S. Dey1,2,3*
Abstract Background: RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification. Results: EMBR-seq results in 90% of the sequenced RNA molecules from an E. coli culture deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq uses a single or a few oligonucleotides per rRNA, thereby making this new technology significantly more costeffective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits for bacterial rRNA depletion, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA. Conclusions: EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples. Keywords: Bacterial mRNA sequencing, mRNA enrichment, rRNA depletion, Low input total RNA
Background Bacterial species pervade our biosphere and millions of years of evolution have optimized these microbes to perform specific biochemical reactions and functions; processes that could potentially be adapted to develop a variety of products, such as renewable * Correspondence: [email protected] † Chatarin Wangsanuwat and Kellie A. Heom contributed equally to this work. 1 Department of Chemical Engineering, University of California Santa Barbara, Santa Barbara, CA 93106, USA 2 Center for Bioengineering, University of California Santa Barbara, Santa Barbara, CA 93106, USA Full list of author information is available at the end of the article
biofuels, antibiotics, and other value-added chemicals [1–5]. Bacterial messenger RNA (mRNA) sequencing provides a snapshot of the genome-wide state of a microbial population, and therefore enables fundamental understanding of these varied microbial functions and phenotypes [6]. However, compared to eukaryotes, mRNA sequencing from bacter
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