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

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.

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis

“…DSN, isolated from the hepatopancreas of the Kamchatka crab (Paralithodes camtschaticus), cleaves double-stranded DNA and RNA-DNA hybrid duplexes, with increased activity on perfectly matched duplexes (Shagin et al 2002). DSN has been used in the normalization of cDNA libraries prior to next generation sequencing (Shagina et al 2010) and the depletion of rRNA from RNA-seq libraries (Christodoulou et al 2011;Yi et al 2011;Matvienko et al 2013;Miller et al 2013). These experiments exploited the knowledge that the rate of DNA hybridization is proportional to the product of the concentration of the two separate DNA strands.…”

“…These experiments exploited the knowledge that the rate of DNA hybridization is proportional to the product of the concentration of the two separate DNA strands. Following denaturation of an RNA-seq cDNA (amplicon) library, the most abundant sequences re-anneal first and can be selectively degraded by addition of DSN (at 68°C), while less abundant sequences remain as ssDNA (Yi et al 2011). To test the effectiveness of DSN in ribosome profiling, we generated Ribo-seq libraries from mouse tissue culture cells and from the green alga Chlamydomonas reinhardtii.…”

Metatranscriptomic Analysis of Arctic Peat Soil Microbiota

“…Several strategies are currently applied to enrich for prokaryotic mRNA molecules. Selective nuclease degradation of rRNA (3)(4)(5), polyadenylation of mRNA (6), and rRNA depletion by capture with commercial kits (3,5,7,8) and sample-specific probes (9) have been attempted to reduce the rRNA fraction of metatranscriptomes.…”

RNA Sequencing Reveals Differences between the Global Transcriptomes of Salmonella enterica Serovar Enteritidis Strains with High and Low Pathogenicities

“…The total-RNA samples were submitted to Illumina, Inc., San Diego, CA, for mRNA enrichment and subsequent RNA-Seq. Removal of 16S and 23S rRNAs from total RNA was performed using a duplex-specific nuclease (DSN) treatment (33). The mRNA was used to prepare individually bar-coded (indexed) RNA-Seq libraries with a TruSeq RNA Sample Prep Kit (Illumina, USA).…”