Probe‐Directed Degradation (PDD) for Flexible Removal of Unwanted cDNA Sequences from RNA‐Seq Libraries. (1st April 2015)

• Record Type: Journal Article
• Authors: Archer, Stuart K.; Shirokikh, Nikolay E.; Preiss, Thomas
• Editors: Haines, Jonathan L.; Korf, Bruce R.; Morton, Cynthia C.; Seidman, Christine E.; Seidman, J.G.; Smith, Douglas R.
• Is Part Of: Current protocols in human genetics. Volume 85(2015)
• Page Start: 11.15.1
• Page End: 11.15.36
• Publication Date: 2015-04-01
• Subjects: RNA‐seq -- removal of rRNA -- cDNA library -- duplex‐specific nuclease -- PDD; Human genetics -- Laboratory manuals; Genetic Techniques; Human genetics; Laboratory manuals; 599.935028
• DOI: 10.1002/0471142905.hg1115s85
• Languages: English
• ISSNs: 1934-8258

Abstract

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. © 2015 by John Wiley & Sons, Inc.