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.
Researchers from the Seoul National University demonstrated the utility of this method to remove rRNA from prokaryotic total RNA 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 the DSN treatment was more efficient at removing rRNA than the Hyb method, while preserving the original relative abundance of mRNA species in bacterial cells.
- 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 Res [Epub ahead of print]. [article]