RNA‐Seq Library Construction Methods for Transcriptome Analysis

Nathan J. Bivens1, Mingyi Zhou1

1 DNA Core Facility, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20019
Online Posting Date:  May, 2016
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Next‐generation sequencing (NGS) technologies have revolutionized the study of genomics with an ever‐expanding list of applications. RNA‐Seq has emerged as a powerful method, applying transcriptome analysis to a wider range of organisms—most significantly, non‐model organisms lacking prior genomic sequencing. Whereas an initial concern of NGS datasets was the potential limitation of short read lengths, short read sequences have been successfully employed in creation of de novo transcriptome assemblies that allow for subsequent mapping of reads for expression analysis. Prior genomic sequence knowledge is no longer a requirement for identification of functional transcriptional elements and for global gene expression characterization. Significant cost reductions in generating RNA‐Seq data, and improvements in de novo assemblers, has allowed the analysis of transcriptomes in heretofore unsequenced plant species. These protocols describe standard methods for constructing RNA‐Seq libraries to be sequenced on Illumina sequencing platforms for comprehensive transcriptome analysis. © 2016 by John Wiley & Sons, Inc.

Keywords: cDNA library; differential gene expression; next‐generation sequencing; RNA‐Seq; transcriptomics

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Table of Contents

  • Introduction
  • Basic Protocol 1: Construction of Stranded (Directional) RNA‐Seq Library by RNA Hydrolysis
  • Alternate Protocol 1: Construction of RNA‐Seq Library with Increased Insert Size
  • Support Protocol 1: DNA Purification Using AxyPrep Mag PCR Clean‐up Beads
  • Commentary
  • Figures
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Basic Protocol 1: Construction of Stranded (Directional) RNA‐Seq Library by RNA Hydrolysis

  • Nuclease Decontamination Solution (to decontaminate surfaces; Integrated DNA Technologies, cat. no. 11‐05‐01‐01)
  • Nuclease‐free water
  • 1 μg total RNA (RNA QC previously performed; see Critical Parameters)
  • TruSeq Stranded mRNA Sample Preparation Kit [Illumina, cat. no. RS‐122‐210(1/2)]
  • SuperScript II Reverse Transcriptase (Invitrogen, cat. no. 18064‐014)
  • Qubit dsDNA HS Assay Kit (Invitrogen, cat. no. Q32851)
  • Standard Sensitivity NGS Fragment Analysis Kit (DNF‐479‐0500)
  • 96‐well nuclease‐free PCR plate with 0.2‐ml well volume
  • Adhesive sealing sheets (Thermo Fisher Scientific, cat. no. AB‐0558)
  • 96‐well thermal cycler (with heated lid)
  • Magnetic stand–96 (Invitrogen, cat. no. 120.27 or Agencourt, part no. A32782)
  • Fluorometer, Qubit (Invitrogen, cat. no. Q32866)
  • Fragment Analyzer (Advanced Analytical Technologies, Inc.)
  • Additional reagents and equipment for DNA purification using the AxyPrep Mag PCR Clean‐up kit ( protocol 3Support Protocol)
NOTE: Use aerosol‐barrier pipet tips and DNA LoBind microcentrifuge tubes in all relevant steps of this protocol.

Alternate Protocol 1: Construction of RNA‐Seq Library with Increased Insert Size

  Additional Materials (also see Basic Protocol 1)
  • Maxima H Minus Double‐Stranded cDNA Synthesis Kit (Thermo Fisher Scientific, cat. no. K2561)
  • 10 to 20 μg total RNA (RNA QC previously performed; see Critical Parameters)
  • TruSeq DNA PCR‐Free Library Prep Kit [Illumina, FC‐122‐300(1/2)]
  • Library Quantification Kit for Illumina Platforms (KAPA Biosystems, cat. no. KK4824)
  • M220 focused ultrasonicator (Covaris, cat. no. M220)
  • MicroTUBE AFA Fiber Screw‐Cap (Covaris, cat. no. 520096)

Support Protocol 1: DNA Purification Using AxyPrep Mag PCR Clean‐up Beads

  • AxyPrep Mag PCR Clean‐up kit (Thermo Fisher Scientific, cat. no. 14‐223‐153)
  • Reaction plate from protocol 1Basic Protocol or protocol 2Alternate Protocol
  • 80% (v/v) ethanol freshly prepared from 200 proof (absolute) ethanol for molecular biology (Sigma‐Aldrich, cat. no. E7023)
  • Magnetic stand–96 (Invitrogen, cat. no. 120.27 or Agencourt, cat. no. A32782)
  • 96‐well nuclease‐free PCR plate with 0.2‐ml well volume
  • Adhesive sealing sheets (Thermo Fisher Scientific, cat. no. AB‐0558)
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Internet Resources
  Web site for Illumina support pages providing access to additional documents and information relating to library methods, library dilution, and sequencing technology referenced in these protocols.
  Wikipedia page providing information on open‐source RNA‐Seq bioinformatics tools.
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