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Abstract BackgroundRNA 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. ResultsEMBR-seq results in 90% of the sequenced RNA molecules from anE. coliculture 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. ConclusionsEMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples.
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This content will become publicly available on March 11, 2026
Duplexed direct RNA sequencing protocol using polyadenylation and polyuridylation
ABSTRACT Oxford Nanopore Technologies provides multiplexing options for DNA and cDNA sequencing, but not for direct RNA sequencing. Here we describe a duplexing approach and validate it by simultaneously sequencing theSaccharomyces cerevisiaerRNA from wild type and knockout that have differential rRNA modifications, successfully demultiplexing the data using bioinformatics approaches.
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- Award ID(s):
- 2025384
- PAR ID:
- 10654436
- Editor(s):
- Roux, Simon
- Publisher / Repository:
- ASM
- Date Published:
- Journal Name:
- Microbiology Resource Announcements
- Volume:
- 14
- Issue:
- 3
- ISSN:
- 2576-098X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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