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dc.contributor.authorPrezza, Gianluca
dc.contributor.authorHeckel, Tobias
dc.contributor.authorDietrich, Sascha
dc.contributor.authorHomberger, Christina
dc.contributor.authorWestermann, Alexander J
dc.contributor.authorVogel, Jörg
dc.date.accessioned2020-08-06T14:07:16Z
dc.date.available2020-08-06T14:07:16Z
dc.date.issued2020-04-28
dc.identifier.citationRNA. 2020;26(8):1069-1078. doi:10.1261/rna.075945.120.en_US
dc.identifier.pmid32345633
dc.identifier.doi10.1261/rna.075945.120
dc.identifier.urihttp://hdl.handle.net/10033/622392
dc.description.abstractA major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative nonribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (depletion of abundant sequences by hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH uses the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56%-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron DASH works robustly, even with subnanogram amounts of input RNA. Its efficiency, high sensitivity, ease of implementation, and low cost (∼$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.en_US
dc.language.isoenen_US
dc.publisherCold Spring Harbor Laboratory Pressen_US
dc.rightsAttribution-NonCommercial-ShareAlike 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.subjectBacteroidesen_US
dc.subjectCRISPRen_US
dc.subjectCas9en_US
dc.subjectDASHen_US
dc.subjectSalmonellaen_US
dc.subjectbacterial RNA-seqen_US
dc.subjectribosomal RNAen_US
dc.titleImproved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads.en_US
dc.typeArticleen_US
dc.identifier.eissn1469-9001
dc.contributor.departmentHIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.en_US
dc.identifier.journalRNA (New York, N.Y.)en_US
dc.source.volume26
dc.source.issue8
dc.source.beginpage1069
dc.source.endpage1078
refterms.dateFOA2020-08-06T14:07:17Z
dc.source.journaltitleRNA (New York, N.Y.)
dc.source.countryUnited States


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