Organism-specific depletion of highly abundant RNA species from bacterial total RNA

Access Microbiology
Florian EngelhardtSusanne Häussler

Abstract

High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93-99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a be...Continue Reading

References

Oct 5, 1990·Journal of Molecular Biology·S F AltschulD J Lipman
Sep 2, 2011·Nucleic Acids Research·Hana YiJongsik Chun
Mar 6, 2012·Nature Methods·Ben Langmead, Steven L Salzberg
May 21, 2013·Nature Methods·Xian AdiconisJoshua Z Levin
Jul 4, 2013·Current Protocols in Molecular Biology·Dominic O'NeilMartin Schlumpberger
Dec 1, 2019·BMC Genomics·Iana V KimClaus-D Kuhn

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Methods Mentioned

BETA
pulldown

Software Mentioned

R script
custom
R
bowtie2
Mcf
Fastq
blast
FastQC

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