Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion
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ABSTRACT:
PROVIDER: PRJNA629575 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11657250_1.fastq.gz | Fastqsanger.gz | |||
| SRR11657250_2.fastq.gz | Fastqsanger.gz | |||
| SRR11657251_1.fastq.gz | Fastqsanger.gz | |||
| SRR11657251_2.fastq.gz | Fastqsanger.gz | |||
| SRR11657252_1.fastq.gz | Fastqsanger.gz |
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Project description: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 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. CONCLUSIONS:EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples.
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