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Volume 2, Issue 10

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

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 , 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 better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.

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  • Accepted:
  • Published Online:
Keyword(s): mRNA enrichment , Pseudomonas aeruginosa , RNA-seq , RNase H , rRNA depletion and tmRNA
Funding
This study was supported by the:
  • Bundesministerium für Bildung und Forschung (Award 03ZZ0816D)
    • Principle Award Recipient: Susanne Häussler
© 2020 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2020-09-09
2024-04-19
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Organism-specific depletion of highly abundant RNA species from bacterial total RNA
Access Microbiology 2, e000159 (2020); https://doi.org/10.1099/acmi.0.000159
/content/journal/acmi/10.1099/acmi.0.000159
/content/journal/acmi/10.1099/acmi.0.000159
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