Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq

Hana Yi, Yong Joon Cho, Sungho Won, Jong Eun Lee, Hyung Jin Yu, Sujin Kim, Gary P. Schroth, Shujun Luo, Jongsik Chun

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

Next-generation sequencing has great potential for application in bacterial transcriptomics. However, unlike eukaryotes, bacteria have no clear mechanism to select mRNAs over rRNAs; therefore, rRNA removal is a critical step in sequencing-based transcriptomics. Duplex-specific nuclease (DSN) is an enzyme that, at high temperatures, degrades duplex DNA in preference to single-stranded DNA. DSN treatment has been successfully used to normalize the relative transcript abundance in mRNA-enriched cDNA libraries from eukaryotic organisms. In this study, we demonstrate the utility of this method to remove rRNA from prokaryotic total RNA. We evaluated the efficacy of DSN to remove rRNA by comparing it with the conventional subtractive hybridization (Hyb) method. Illumina deep sequencing was performed to obtain transcriptomes from Escherichia coli grown under four growth conditions. The results clearly showed that our DSN treatment was more efficient at removing rRNA than the Hyb method was, while preserving the original relative abundance of mRNA species in bacterial cells. Therefore, we propose that, for bacterial mRNA-seq experiments, DSN treatment should be preferred to Hyb-based methods.

Original languageEnglish
JournalNucleic Acids Research
Volume39
Issue number20
DOIs
Publication statusPublished - 2011 Nov 1
Externally publishedYes

Fingerprint

RNA
Messenger RNA
High-Throughput Nucleotide Sequencing
Single-Stranded DNA
Eukaryota
Gene Library
Transcriptome
Escherichia coli
Bacteria
Temperature
DNA
Enzymes
Growth
Experiments
Subtractive Hybridization Techniques

ASJC Scopus subject areas

  • Genetics

Cite this

Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq. / Yi, Hana; Cho, Yong Joon; Won, Sungho; Lee, Jong Eun; Jin Yu, Hyung; Kim, Sujin; Schroth, Gary P.; Luo, Shujun; Chun, Jongsik.

In: Nucleic Acids Research, Vol. 39, No. 20, 01.11.2011.

Research output: Contribution to journalArticle

Yi, H, Cho, YJ, Won, S, Lee, JE, Jin Yu, H, Kim, S, Schroth, GP, Luo, S & Chun, J 2011, 'Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq', Nucleic Acids Research, vol. 39, no. 20. https://doi.org/10.1093/nar/gkr617
Yi, Hana ; Cho, Yong Joon ; Won, Sungho ; Lee, Jong Eun ; Jin Yu, Hyung ; Kim, Sujin ; Schroth, Gary P. ; Luo, Shujun ; Chun, Jongsik. / Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq. In: Nucleic Acids Research. 2011 ; Vol. 39, No. 20.
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