Next‐Generation Sequencing RNA‐Seq Library Construction

Jessica Podnar1, Heather Deiderick1, Gabriella Huerta1, Scott Hunicke‐Smith1

1 Genomic Sequencing and Analysis Facility, University of Texas at Austin, Austin, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.21
DOI:  10.1002/0471142727.mb0421s106
Online Posting Date:  April, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit presents protocols for construction of next‐generation sequencing (NGS) directional RNA sequencing libraries for the Illumina HiSeq and MiSeq from a wide variety of input RNA sources. The protocols are based on the New England Biolabs (NEB) small RNA library preparation set for Illumina, although similar kits exist from different vendors. The protocol preserves the orientation of the original RNA in the final sequencing library, enabling strand‐specific analysis of the resulting data. These libraries have been used for differential gene expression analysis and small RNA discovery and are currently being tested for de novo transcriptome assembly. The protocol is robust and applicable to a broad range of RNA input types and RNA quality, making it ideal for high‐throughput laboratories. Curr. Protoc. Mol. Biol. 106:4.21.1‐4.21.19. © 2014 by John Wiley & Sons, Inc.

Keywords: RNA‐Seq; NGS; library construction; transcriptome; strand‐specific; gene expression

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: RNA Fragmentation
  • Basic Protocol 2: Library Construction
  • Basic Protocol 3: Size Selection with AMPure Beads
  • Basic Protocol 4: Size Selection with AMPure XP Beads (Small RNA Libraries)
  • Basic Protocol 5: Alternative Size Selection for Small RNA Libraries (Blue Pippin Prep)
  • Post‐Sequencing Bioinformatics Protocols
  • Support Protocol 1: Estimation of Adaptor‐Dimer, Short‐Product, or “No Insert” Contamination
  • Support Protocol 2: Estimation of Library Size Distribution if Less than Read Length
  • Support Protocol 3: Finding Unexplained “Dominant Sequences”
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: RNA Fragmentation

  Materials
  • Purified RNA (2 to 50 µg total RNA or 50 to 250 ng mRNA; see Chapter 4)
  • NEBNext Magnesium RNA Fragmentation Module (NEB, cat. no. E6150S) containing:
    • 10× RNA Fragmentation Buffer
    • Nuclease‐free water
    • 10× RNA Fragmentation Stop Solution
  • RNeasy MinElute Cleanup Kit (Qiagen, cat. no. 74204)
  • T4 Polynucleotide Kinase Kit (NEB, cat. no. M0201), including:
    • T4 polynucleotide kinase (PNK)
    • T4 polynucleotode kinase (PNK) buffer
  • 10 mM ATP (Invitrogen, cat. no. AM8110G)
  • RiboMinus Concentration Module (Invitrogen, cat. no. K155005) including:
    • Binding buffer
    • 100% ethanol
    • Wash buffer (W5)
  • Thermal cycler

Basic Protocol 2: Library Construction

  Materials
  • RNA (100 ng 1 µg typically required; e.g., from protocol 1)
  • NEBNext Small RNA Library Prep Set for Illumina (Multiplex Compatible; NEB, cat. no. E7300; comes with barcodes) including:
    • Nuclease‐free water
    • 3′ SR Adaptor for Illumina
    • 3′ Ligation Reaction Buffer (2×)
    • 3′ Ligation Enzyme Mix
    • SR RT Primer for Illumina
    • 5′ SR Adaptor for Illumina
    • 5′ Ligation Reaction Buffer (10×)
    • 5′ Ligase Enzyme Mix
    • Deoxynucleotide Solution Mix
    • Murine RNase inhibitor
    • Long Amp Taq 2× Master Mix
    • Multiplex SR primer
    • Index (X) Primer‐Index 1‐12
  • SuperScript III Reverse Transcriptase (Invitrogen, cat. no. 18080‐044)
  • 10 mM dNTP mix (NEB; cat no. N0447L)
  • 5× First Strand Buffer (supplied with SuperScript III)
  • 0.1 M DTT (supplied with SuperScript III)
  • Thermal cycler
  • Additional reagents and equipment for purification/size selection ( protocol 3, 4, or 5)

Basic Protocol 3: Size Selection with AMPure Beads

  Materials
  • Agencourt AMPure XP beads (Beckman Coulter, cat. no. NCC9933872)
  • Sample (see protocol 3, step 24)
  • 80% ethanol (prepared with nuclease‐free H 2O)
  • qPCR standards: Kapa Biosystems Universal (cat no. KK4602) and standards (cat. no. KK4903)
  • Calibrated fluorescence assay kit, e.g., Picogreen or Qubit (optional; Life Technologies)
  • Magnetic rack (Ambion, cat. no. AM10055)
  • Nuclease‐free water
  • Agilent Bioanalyzer and appropriate DNA chip
  • Additional reagents and equipment for quantitative PCR (unit )

Basic Protocol 4: Size Selection with AMPure XP Beads (Small RNA Libraries)

  Materials
  • Agencourt AMPure XP beads (Beckman Coulter, cat. no. NCC9933872)
  • Sample (see protocol 3, step 24)
  • 80% ethanol (prepared using nuclease‐free H 2O)
  • qPCR standards: Kapa Biosystems Universal (cat no. KK4602) and standards (cat. no. KK4903)
  • Calibrated fluorescence assay kit, e.g., Picogreen or Qubit (optional; Life Technologies)
  • Magnetic rack (Ambion, cat. no. AM10055)
  • Nuclease‐free water
  • Agilent Bioanalyzer and appropriate DNA chip
  • Additional reagents and equipment for quantitative PCR (unit )

Basic Protocol 5: Alternative Size Selection for Small RNA Libraries (Blue Pippin Prep)

  Materials
  • BluePippin DNA Size Selection System (Sage Science, cat. no. BLU0001)
  • 2% BluePippin Agarose Gel Cassettes, Dye‐free (Sage Science, cat. no. BDF2010)
  • BluePippin V1 Marker Mix/Loading Buffer
  • BluePippin Electrophoresis Buffer
  • TE Buffer ( )
  • MinElute PCR Purification Kit, Qiagen #28004
  • 2% casettes
  • Adhesive plate covers
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Birol, I. , Jackman, S.D. , Nielsen, C.B. , Qian, J.Q. , Varhol, R. , Stazyk, G. , Morin, R.D. , Zhao, Y. , Hirst, M. , Schein, J.E. , Horsman, D.E. , Connors, J.M. , Gascoyne, R.D. , Marra, M.A. , and Jones, S.J. 2009. De novo transcriptome assembly with ABySS. Bioinformatics 25:2872‐2877.
   Chu, Y. and Corey, D.R. 2012. RNA sequencing: Platform selection, experimental design, and data interpretation. Nucleic Acid Ther. 22:271‐274.
   Cloonan, N. , Forrest, A.R. , Kolle, G. , Gardiner, B.B. , Faulkner, G.J. , Brown, M.K. , Taylor, D.F. , Steptoe, A.L. , Wani, S. , Bethel, G. , Robertson, A.J. , Perkins, A.C. , Bruce, S.J. , Lee, C.C. , Ranade, S.S. , Peckham, H.E. , Manning, J.M. , McKernan, K.J. , and Grimmond, S.M. 2008. Stem cell transcriptome profiling via massive‐scale mRNA sequencing. Nat. Methods 5:613‐619.
   Djebali, S. , Davis, C.A. , Merkel, A. , Dobin, A. , Lassmann, T. , Mortazavi, A. , Tanzer, A. , Lagarde, J. , Lin, W. , Schlesinger, F. , Xue, C. , Marinov, G.K. , Khatun, J. , Williams, B.A. , Zaleski, C. , Rozowsky, J. , Röder, M. , Kokocinski, F. , Abdelhamid, R.F. , Alioto, T. , Antoshechkin, I. , Baer, M.T. , Bar, N.S. , Batut, P. , Bell, K. , Bell I. , Chakrabortty, S. , Chen, X. , Chrast, J. , Curado, J. , Derrien, T. , Drenkow, J. , Dumais, E. , Dumais, J. , Duttagupta, R. , Falconnet, E. , Fastuca, M. , Fejes‐Toth, K. , Ferreira, P. , Foissac, S. , Fullwood, M.J. , Gao, H. , Gonzalez, D. , Gordon, A. , Gunawardena, H. , Howald, C. , Jha, S. , Johnson, R. , Kapranov, P. , King, B. , Kingswood, C. , Luo, O.J. , Park, E. , Persaud, K. , Preall, J.B. , Ribeca, P. , Risk, B. , Robyr, D. , Sammeth, M. , Schaffer, L. , See, L.H. , Shahab, A. , Skancke, J. , Suzuki, A.M. , Takahashi, H. , Tilgner, H. , Trout, D. , Walters, N. , Wang, H. , Wrobel, J. , Yu, Y. , Ruan, X. , Hayashizaki, Y. , Harrow, J. , Gerstein, M. , Hubbard, T. , Reymond, A. , Antonarakis, S.E. , Hannon, G. , Giddings, M.C. , Ruan, Y. , Wold, B. , Carninci, P. , Guigó, R. , and Gingeras, T.R. 2012. Landscape of transcription in human cells. Nature 489:101‐108.
   Haas, B.J. and Zody, M.C. 2010. Advancing RNA‐Seq analysis. Nat. Biotechnol. 28:421‐423.
   Levin, J.Z. , Yassour, M. , Adiconis, X. , Nusbaum, C. , Thompson, D.A. , Friedman, N. , Gnirke, A. , and Regev, A. 2010. Comprehensive comparative analysis of strand‐specific RNA sequencing methods. Nat. Methods 7:709‐715.
   Linsen, S.E. , de Wit, E. , Janssens, G. , Heater, S. , Chapman, L. , Parkin, R.K. , Fritz, B. , Wyman, S.K. , de Bruijn, E. , Voest, E.E. , Kuersten, S. , Tewari, M. , and Cuppen, E. 2009. Limitations and possibilities of small RNA digital gene expression profiling. Nat. Methods 6:474‐476.
   Liu, J. , Jennings, S.F. , Tong, W. , and Hong, H. 2011. Next generation sequencing for profiling expression of miRNAs: Technical progress and applications in drug development. J. Biomed. Sci. Eng. 4:666‐676.
   Lu, C. , Tej, S.S. , Luo, S. , Haudenschild, C.D. , Meyers, B.C. , and Green, P.J. 2005. Elucidation of the small RNA component of the transcriptome. Science 309:1567‐1569.
   Mamanova, L. , Andrews, R.M. , James, K.D. , Sheridan, E.M. , Ellis, P.D. , Langford, C.F. , Ost, T.W. , Collins, J.E. , and Turner, D.J. 2010. FRT‐seq: Amplification‐free, strand‐specific, transcriptome sequencing. Nat. Methods 7:130‐132.
   Morin, R. , Bainbridge, M. , Fejes, A. , Hirst, M. , Krzywinski, M. , Pugh, T. , McDonald, H. , Varhol, R. , Jones, S. , and Marra, M. 2008. Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short‐read sequencing. Biotechniques 45:81‐94.
   Mortazavi, A. , Williams, B.A. , McCue, K. , Schaeffer, L. , and Wold, B. 2008. Mapping and quantifying mammalian transcriptomes by RNA‐Seq. Nat. Methods 5:621‐628.
   Nagalakshmi, U. , Wang, Z. , Waern, K. , Shou, C. , Raha, D. , Gerstein, M. , and Snyder, M. 2008. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320:1344‐1349.
   Reid, J.G. , Nagaraja, A.K. , Lynn, F.C. , Drabek, R.B. , Muzny, D.M. , Shaw, C.A. , Weiss, M.K. , Naghavi, A.O. , Khan, M. , Zhu, H. , Tennakoon, J. , Gunaratne, G.H. , Corry, D.B. , Miller, J. , McManus, M.T. , German, M.S. , Gibbs, R.A. , Matzuk, M.M. , and Gunaratne, P.H. 2008. Mouse let‐7 miRNA populations exhibit RNA editing that is constrained in the 5′‐seed/cleavage/anchor regions and stabilize predicted mmu‐let‐7a:mRNA duplexes. Genome Res. 18:1571‐1581.
   Rozowsky, J. , Abyzov, A. , Wang, J. , Alves, P. , Raha, D. , Harmanci, A. , Leng, J. , Bjornson, R. , Kong, Y. , Kitabayashi, N. , Bhardwaj, N. , Rubin, M. , Snyder, M. , and Gerstein, M. 2011. AlleleSeq: Analysis of allele‐specific expression and binding in a network framework. Mol. Syst. Biol. 7:522.
   Trapnell, C. , Williams, B.A. , Pertea, G. , Mortazavi, A. , Kwan, G. , van Baren, M.J. , Salzberg, S.L. , Wold, B.J. , and Pachter, L. 2010. Transcript assembly and quantification by RNA‐Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28:511‐515.
   Wang, Z. , Gerstein, M. , and Snyder, M. 2009. RNA‐Seq: A revolutionary tool for transcriptomics. Nat. Rev. Genet. 10:57‐63.
   Wilhelm, B.T. , Marguerat, S. , Watt, S. , Schubert, F. , Wood, V. , Goodhead, I. , Penkett, C.J. , Rogers, J. , and Bähler, J. 2008. Dynamic repertoire of a eukaryotic transcriptome surveyed at single‐nucleotide resolution. Nature 453:1239‐1243.
   Zhuang, F. , Fuchs, R.T. , Sun, Z. , Zheng, Y. , and Robb, G.B. 2012. Structural bias in T4 RNA ligase‐mediated 3′‐adapter ligation. Nucleic Acids Res. 40:e54.
Internet Resources
   http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
  This site provides a free tool, FastQC, which can be used to assess DNA sequences from the Illumina platform. It is not particularly well suited to RNA analysis because it contains built‐in metrics that were derived from whole genome DNA sequencing, but is nonetheless a benchmark tool.
   http://www.chem.agilent.com/Library/eseminars/Public/03012011_eSeminar_Bioanalyzer%20Apps%20and%20Tips%20for%20NGS_v2.pdf
  Provides helpful benchmark data such as bead carry‐over in Agilent BioAnalyzer, and other suggestions on NGS library assessment.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library