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, researchers from the University of California Santa Barbara have developed 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.
EMBR-seq results in 90% of the sequenced RNA molecules from an E. coli culture deriving from mRNA. The researchers 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 cost-effective, 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.
Schematic of EMBR-Seq
After performing an optional Terminator™ 5′-phosphate-dependent exonuclease digestion, poly-A polymerase and rRNA blocking primers (purple) are added to total bacterial RNA (mRNA in red and rRNA in gray). Blocking primers specifically bind to the 3′ end of 5S, 16S, and 23S rRNAs, resulting in the preferential addition of a poly-A tail to mRNA molecules. Next, reverse transcription is performed using (i) a poly-T primer, which has an overhang containing a sample-specific barcode (blue), 5′ Illumina adapter (yellow), and T7 promoter (green), and (ii) rRNA blocking primers to convert poly-adenylated RNA and rRNA molecules, respectively, to cDNA. The cDNA molecules are then amplified by in vitro transcription, and the amplified RNA is used to prepare Illumina libraries. As the rRNA-derived cDNA does not contain a T7 promoter, these molecules are not amplified during in vitro transcription, resulting in rRNA depletion