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

Content Provider	Springer Nature : BioMed Central
Author	Wangsanuwat, Chatarin Heom, Kellie A. Liu, Estella O’Malley, Michelle A. Dey, Siddharth S.
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 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.
Related Links	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-020-07134-4.pdf
Ending Page	12
Page Count	12
Starting Page	1
File Format	HTM / HTML
ISSN	14712164
DOI	10.1186/s12864-020-07134-4
Journal	BMC Genomics
Issue Number	1
Volume Number	21
Language	English
Publisher	BioMed Central
Publisher Date	2020-10-16
Access Restriction	Open
Subject Keyword	Life Sciences Microarrays Proteomics Animal Genetics and Genomics Microbial Genetics and Genomics Plant Genetics and Genomics Bacterial mRNA sequencing mRNA enrichment rRNA depletion Low input total RNA
Content Type	Text
Resource Type	Article
Subject	Biotechnology Genetics
Journal Impact Factor	3.5/2023
5-Year Journal Impact Factor	4.1/2023

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