title: Probe-Directed Degradation (PDD) for Flexible Removal of Unwanted cDNA Sequences from RNA-Seq Libraries.
abstract: Most applications for RNA-seq require the depletion of abundant transcripts to gain greater coverage of the underlying transcriptome. The sequences to be targeted for depletion depend on application and species and in many cases may not be supported by commercial depletion kits. This unit describes a method for generating RNA-seq libraries that incorporates probe-directed degradation (PDD), which can deplete any unwanted sequence set, with the low-bias split-adapter method of library generation (although many other library generation methods are in principle compatible). The overall strategy is suitable for applications requiring customized sequence depletion or where faithful representation of fragment ends and lack of sequence bias is paramount. We provide guidelines to rapidly design specific probes against the target sequence, and a detailed protocol for library generation using the split-adapter method including several strategies for streamlining the technique and reducing adapter dimer content.
authors.affiliation: Genome Biology Department, The John Curtin School of Medical Research (JCSMR), The Australian National University, Acton, Canberra, Australian Capital Territory, Australia.|Present address: Monash Bioinformatics Platform, Monash University, Clayton, Victoria, Australia.|These authors contributed equally to this work.
authors.forename: Stuart K
authors.initials: SK
authors.lastname: Archer
authors.affiliation: Genome Biology Department, The John Curtin School of Medical Research (JCSMR), The Australian National University, Acton, Canberra, Australian Capital Territory, Australia.|Present address: Moscow Regional State Institute of Humanities and Social Studies, Ministry of Education of Moscow Region, Kolomna, Moscow Region, Russia.|These authors contributed equally to this work.
authors.forename: Nikolay E
authors.initials: NE
authors.lastname: Shirokikh
authors.affiliation: Genome Biology Department, The John Curtin School of Medical Research (JCSMR), The Australian National University, Acton, Canberra, Australian Capital Territory, Australia.|Victor Chang Cardiac Research Institute, Darlinghurst (Sydney), New South Wales, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Preiss
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism*; D004260:DNA Repair; D004270:DNA, Circular / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D015723:Gene Library; D005821:Genetic Techniques; D026723:Polyadenylation; D020871:RNA Stability; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D017423:Sequence Analysis, RNA
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D004270:DNA, Circular; D018076:DNA, Complementary; D012259:Ribonuclease, Pancreatic
keywords: PDD; RNA-seq; cDNA library; duplex-specific nuclease; removal of rRNA