Normalization of Genomic DNA Using Duplex-Specific Nuclease

Shagina, Irina; Богданова, Е. А.; Mamedov, Ilgar Z.; Lebedev, Yuri B.; Lukyanov, Sergey; Shagin, Dmitry A.

doi:10.2144/000113422

Cited by 45 publications

(40 citation statements)

References 25 publications

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“…Additional RNA-Seq data sets were generated from these RNA samples after treatment with the DSN (Duplex-Specific Nuclease) method to deplete rRNA sequences [38]. As expected, most of the DSN-treated samples include a higher fraction of reads derived from non-rRNA sequences (Table S1) and show the strongest correlation with the corresponding RNA-Seq data sets obtained from Total RNA ( e.g ., NSM-DSN vs NSM total RNA, Fig.…”

Section: Resultsmentioning

confidence: 71%

Isolation of Specific Neurons from C. elegans Larvae for Gene Expression Profiling

et al. 2014

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BackgroundThe simple and well-described structure of the C. elegans nervous system offers an unprecedented opportunity to identify the genetic programs that define the connectivity and function of individual neurons and their circuits. A correspondingly precise gene expression map of C. elegans neurons would facilitate the application of genetic methods toward this goal. Here we describe a powerful new approach, SeqCeL (RNA-Seq of C. elegans cells) for producing gene expression profiles of specific larval C. elegans neurons.Methods and ResultsWe have exploited available GFP reporter lines for FACS isolation of specific larval C. elegans neurons for RNA-Seq analysis. Our analysis showed that diverse classes of neurons are accessible to this approach. To demonstrate the applicability of this strategy to rare neuron types, we generated RNA-Seq profiles of the NSM serotonergic neurons that occur as a single bilateral pair of cells in the C. elegans pharynx. These data detected >1,000 NSM enriched transcripts, including the majority of previously known NSM-expressed genes.SignificanceThis work offers a simple and robust protocol for expression profiling studies of post-embryonic C. elegans neurons and thus provides an important new method for identifying candidate genes for key roles in neuron-specific development and function.

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Section: Resultsmentioning

confidence: 71%

Isolation of Specific Neurons from C. elegans Larvae for Gene Expression Profiling

et al. 2014

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Chung

Hardcastle

Jones

et al. 2015

RNA

120

132

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Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.

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“…Email: endeldw @ uc.edu DNA has also been applied for many techniques using short RNA sequences [16,[18][19][20][21][22][23][24][25][26][27][28][29], but applications for long RNA targets have remained elusive.…”

Section: * Corresponding Authormentioning

confidence: 99%

Rational Design of Duplex Specific Nuclease for One-Step Isothermal Viral RNA Detection

2017

J App Biol Biotech

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RNA viruses are a potent human adversary, evidenced by several global pandemics including the Ebolavirus in West Africa, the emerging Zika virus, and outbreaks of new Influenza strains and Norwalk virus in the food supply and cruise ships. Despite the virulence of these pathogens, there remains a significant limitation for detecting these viruses in a fast, accurate and cost effective manner. To meet this need we present a modified form of the duplex specific nuclease enzyme from the Paralithodes camtschaticus crab capable of generating an RNA-based signal amplification in a fraction of the time required for standard RT-qPCR. The applicability of this enzyme is demonstrated in an assay for Norwalk virus detection with a lower limit of ~100 viral copies per liter of environmental water.

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Normalization of Genomic DNA Using Duplex-Specific Nuclease

Cited by 45 publications

References 25 publications

Isolation of Specific Neurons from C. elegans Larvae for Gene Expression Profiling

Isolation of Specific Neurons from C. elegans Larvae for Gene Expression Profiling

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

Rational Design of Duplex Specific Nuclease for One-Step Isothermal Viral RNA Detection

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