Complete Transcriptome RNA-Seq

David F.B. Miller, Pearlly Yan, Fang Fang, Aaron Buechlein, Karl Kroll, David Frankhouser, Cameron Stump, Paige Stump, James B. Ford, Haixu Tang, Scott Michaels, Daniela Matei, Tim H. Huang, Jeremy Chien, Yunlong Liu, Douglas B. Rusch, Kenneth P. Nephew

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

RNA-Seq is the leading technology for analyzing gene expression on a global scale across a broad spectrum of sample types. However, due to chemical modifi cations by fi xation or degradation due to collection methods, samples often contain an abundance of RNA that is no longer intact, and the capability of current RNA-Seq protocols to accurately quantify such samples is often limited. We have developed an RNASeq protocol to address these key issues as well as quantify gene expression from the whole transcriptome. Furthermore, for compatibility with improved sequencing platforms, we use restructured adapter sequences to generate libraries for Illumina HiSeq, MiSeq, and NextSeq platforms. Our protocol utilizes duplexspecifi c nuclease (DSN) to remove abundant ribosomal RNA sequences while retaining other types of RNA for superior transcriptome profi ling from low quantity input. We employ the Illumina sequencing platform, but this method is described in suffi cient detail to adapt to other platforms.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Pages141-162
Number of pages22
DOIs
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1513
ISSN (Print)1064-3745

Keywords

  • Duplex-specifi c Nuclease
  • Gene Expression
  • RNA-Seq
  • Sequencing
  • Transcriptome

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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