Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

Blog Article

Abstract Background RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population click here 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 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 el reformador tequila anejo 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.