Stranded Whole Transcriptome RNA‐Seq for All RNA Types

David F.B. Miller1, Pearlly X. Yan2, Fang Fang1, Aaron Buechlein3, James B. Ford3, Haixu Tang3, Tim H. Huang4, Matthew E. Burow5, Yunlong Liu6, Douglas B. Rusch3, Kenneth P. Nephew1

1 Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana, 2 Department of Internal Medicine, OSUCCC‐Illumina Core, Columbus, Ohio, 3 Indiana University Center for Genomics and Bioinformatics, Bloomington, Indiana, 4 Department of Molecular Medicine, University of Texan Health Science Center, San Antonio, Texas, 5 Department of Medicine, Tulane University, New Orleans, Louisiana, 6 Indiana University School of Medicine, Center for Computation Biology and Bioinformatics, Indianapolis, Indiana
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 11.14
DOI:  10.1002/0471142905.hg1114s84
Online Posting Date:  January, 2015
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Abstract

Stranded whole transcriptome RNA‐Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to, miRNA (microRNA), piRNA (Piwi‐interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non‐coding intergenic RNA), SRP RNA (signal recognition particle RNA), tRNA (transfer RNA), mtRNA (mitochondrial RNA), and mRNA (messenger RNA). The size and nature of these types of RNA are irrelevant to the approach described here. Barcoded libraries for multiplexing on the Illumina platform are generated with this approach but it can be applied to other platforms with a few modifications. © 2015 by John Wiley & Sons, Inc.

Keywords: RNA‐Seq; transcriptome; gene expression; duplex‐specific nuclease

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: RNA Purification and Fragmentation
  • Basic Protocol 2: End Repair and Adapter Ligations
  • Basic Protocol 3: Reverse Transcription and PCR1
  • Support Protocol 1: Ampure XP Calibration and Clean‐Up for PCR1 Reaction
  • Basic Protocol 4: Duplex‐Specific Nuclease (DSN) Reduction of rRNA Sequences
  • Basic Protocol 5: PCR2 Amplification and Barcode Addition of DSN Normalized Libraries
  • Support Protocol 2: qRT‐PCR Validation of DSN rRNA Sequence Reduction
  • Basic Protocol 6: Quantification and Pooling Barcoded Libraries for Sequencing on the Illumina Platform
  • Adapting This Approach to Other Adapters
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: RNA Purification and Fragmentation

  Materials
  • RLT buffer (Qiagen)
  • 2‐Mercaptoethanol (BME)
  • Tissue culture cells or other suitable source for RNA
  • RNeasy Mini AllPrep kit (Qiagen) containing:
    • AllPrep AP DNA column
    • RPE buffer
  • 100 % (v/v) Ethanol
  • RNeasy Minelute kit (Qiagen) containing:
    • RNeasy column
  • RWT buffer (Qiagen)
  • Nuclease‐free water (Sigma)
  • 10× fragmentation reagent and stop solution (Ambion)
  • RNA 6000 Pico assay kit (Agilent)
  • 1.7‐ml tubes (Avant)
  • Vortex mixer
  • Qiashredder column (Qiagen)
  • Microcentrifuge
  • NanoDrop 2000 (ThermoFisher)
  • 2100 Bioanalyzer (Agilent)

Basic Protocol 2: End Repair and Adapter Ligations

  Materials
  • smRNA or FLgRNA in 17 μl RNase‐free water (see Basic Protocol 1)
  • Antarctic phosphatase (New England Biolabs) containing:
    • Antarctic phosphatase reaction buffer
  • RNasin Plus (Promega)
  • Ice
  • T4 polynucleotide kinase containing:
    • T4 polynucleotide kinase buffer
  • Nuclease‐free water (Sigma)
  • RNeasy Minelute kit (Qiagen) containing:
    • RNeasy Minelute columns
  • RS‐TS 3′ adapter (5 μM) (Integrated DNA Technologies)
  • T4 RNA Ligase 2, truncated KQ (New England Biolabs) containing:
    • T4 RNA ligase 2, truncated KQ buffer
  • RS‐TS‐PCR1A primer (5 μM) (Integrated DNA Technologies)
  • RT‐TS 5′ adapter (10 μM)
  • T4 RNA Ligase 1 (New England Biolabs) containing:
    • T4 RNA ligase 1 reaction buffer
  • 0.2‐ml PCR tubes
  • Thermal cycler with a heated lid
  • NanoDrop 2000 (ThermoFisher)

Basic Protocol 3: Reverse Transcription and PCR1

  Materials
  • Adapter‐ligated RNA in 10 μl RNase‐free water (see Basic Protocol 2)
  • RS‐TS PCR 1A primer (25 μM)
  • Superscript RT III (Life Technologies, cat. no. 18080‐400)
  • Superscript RT III/RNaseOut enzyme mixture (supplied with the Superscript RT kit by Life Technologies)
  • Superscript 2X reaction buffer (supplied with kit)
  • RNase H (Life Technologies)
  • RS‐TS PCR 2D primer (25 μM)
  • 40 mM EDTA, pH8
  • 100 mM dNTP mix (Bioline)
  • Phusion DNA polymerase (New England Biolabs) containing:
    • Phusion reaction buffer (supplied with Phusion DNA polymerase)
  • RNase‐free water
  • 0.2‐ml PCR tubes
  • Thermal cycler with a heated lid

Support Protocol 1: Ampure XP Calibration and Clean‐Up for PCR1 Reaction

  Materials
  • 20‐bp Ext Range DNA Ladder (Lonza)
  • EB elution buffer (Qiagen)
  • Tween 20 (Promega)
  • AMPure XP magnetic beads (Beckman Coulter)
  • 100% ethanol
  • Nuclease‐free water (Sigma)
  • Bioanalyzer HS DNA kit (Agilent)
  • 1.7‐ml tubes
  • Vortex mixer
  • Magna‐Sep magnetic 1.7‐ml tube stand (Life Technologies)
  • 2100 Bioanalyzer (Agilent)

Basic Protocol 4: Duplex‐Specific Nuclease (DSN) Reduction of rRNA Sequences

  Materials
  • PCR1 library DNA
  • Custom 10× DSN hybridization buffer (see recipe)
  • Nuclease‐free water (Sigma)
  • 2× custom DSN reaction buffer (see recipe)
  • Duplex‐specific nuclease (Wako Chemicals)
  • 2× custom DSN stop buffer (see recipe)
  • Ice
  • AMPure XP beads (Beckman Coulter)
  • EB elution buffer (Qiagen; same as provided in the AllPrep kit)
  • 0.2‐μl PCR tubes
  • Centrifuge
  • Thermal cycler (2 chamber or additional thermal cycler)
  • 1.7‐ml tubes

Basic Protocol 5: PCR2 Amplification and Barcode Addition of DSN Normalized Libraries

  Materials
  • DSN normalized library (see Basic Protocol 5)
  • Phusion DNA polymerase (New England Biolabs) containing:
    • 5× Phusion reaction buffer (supplied with Phusion polymerase)
  • RS‐TS PCRI‐# barcoded primer (25 μM)
  • RS‐TS PCR2D primer (25 μM)
  • 100 mM dNTP mix (Bioline)
  • Nuclease‐free water
  • AMPure XP beads (Beckman Coulter)
  • EB elution buffer (Qiagen)
  • Tween 20
  • Qubit dsDNA HS assay (Life Technologies)
  • EB buffer containing 0.1% Tween 20 (EBT)
  • 0.2‐ml PCR tubes
  • Thermal cycler with a heated lid
  • Bioanalyzer HS DNA kit (Agilent)
  • Additional reagents and equipment for clean up of the PCR2 barcoded libraries with AMPure XP beads (see Support Protocol 1)

Support Protocol 2: qRT‐PCR Validation of DSN rRNA Sequence Reduction

  Materials
  • PCR1 library (see Basic Protocol 3 and Support Protocol 1)
  • PCR2 library (see Basic Protocol 5)
  • EB buffer (Qiagen)
  • Tween 20 (Promega)
  • 5S rRNA PCR primers (5 μl each of forward and reverse primers in nuclease‐free water) (ThermoFisher)
  • 28S rRNA PCR primers (5 μl each of forward and reverse primers in nuclease‐free water) (ThermoFisher)
  • EEF1A1 PCR primers (5 μl each of forward and reverse primers in nuclease‐free water) (ThermoFisher)
  • U6 snRNA PCR primers (ThermoFisher)
  • Nuclease‐free water
  • LightCycler 480 SYBR Green I Master mix (Roche)
  • 480 LightCycler 96‐well plates (Roche)
  • 480 LightCycler (Roche)

Basic Protocol 6: Quantification and Pooling Barcoded Libraries for Sequencing on the Illumina Platform

  Materials
  • Libraries to be pooled
  • Qubit dsDNA HS assay (Life Technologies)
  • EB buffer (Qiagen)
  • Tween 20 (Promega)
  • Bioanalyzer HS DNA kit (Agilent)
  • 2100 Bioanalyzer (Agilent)
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Figures

Videos

Literature Cited

Literature Cited
  Livak, K. and Schmitgen, T. 2001. Analysis of relative gene expression data using real‐time quantitative PCR and the 2‐ΔΔCT method. Methods 25:402‐408.
  McGettigan, P. 2012. Transcriptomics in the RNA‐seq era. Curr. Opin. Chem. Biol. 17:1‐8.
  Morlan, J., Qu, K., and Sinicropi, D. 2012. Selective depletion of rRNA enables whole transcriptome profiling of Archival fixed tissue. Plos One 7:1‐8.
  Nagalakshmi, U., Wang, Z., Waern, K., Shou, C., Raha, D., Gerstein, M., and Snyder, M. 2008. Comprehensive analysis of RNA‐seq data reveals extensive RNA editing in a human transcriptome. Science 320:1344‐1349.
  Peng, Z., Cheng, Y., Tan, B., Kang, L., Tian, Z., Zhu, Y., Zhang, W., Liang, Y., Hu, X., Tan, X., Guo, J., Dong, Z., Liang, Y., Bao, L., and Wang, J. 2012. Comprehensive analysis of RNA‐seq data reveals extensive RNA editing in a human transcriptome. Nat. Biotech. 30:253‐262.
  Strong, A., Shi, Z., Strong, M., Miller, D., Rusch, D., Buechlein, A., Flemington, E., McLachlan, J., Nephew, K., Burow, M., and Bunnell, B. 2014. Effects of the endocrine‐disrupting chemical DDT on self‐renewal and differentiation of human mesenchymal stem cells. Environ. Health Perspect. Epub ahead of print: DOI:10.1289/ehp.1408188
  Vigneault, F., Ter‐Ovanesyan, D., Alon, S., Eminaga, S. C., Christodoulou, D., Seidman, J. G., Eisenberg, E. M. and Church, G. 2012. High‐Throughput Multiplex Sequencing of miRNA. Curr. Protoc. Hum. Genet. 73:11.12:11.12.1–11.12.10.
  Yoffe, A., Prinsen, P., Gopal, A., Knobler, C., Gelbart, W., and Ben‐Shaul, A. 2008. Prediction the sizes of large RNA molecules. Proc. Natl. Acad. Sci. U.S.A. 105:16153‐16158.
Key References
  Miller, D., Yan, P., Buechlein, A., Rodriguez, B., Yilmaz, A., Goel, S., Lin, H., Collins‐Burow, B., Rhodes, L., Braun, C., Pradeep, S., Rupaimoole, R., Dalkilic, M., Sood, A., Burow, M., Tang, H., Huang, T., Liu, Y., Rusch, D., and Nephew, K. 2013. A new method for stranded whole transcriptome RNA‐seq. Methods 63:126‐134.
  This original publication for the method in this unit is described in this publication. The protocol in this unit has been updated with improvements.
Internet Resources
  http://www.idtdna.com/analyzer/applications/oligoanalyzer/
  IDT (Integrated DNA Technology) OligoAnalyzer 3.1.
  http://support.illumina.com/downloads/multiplexing_sample_prep_guide_1005361.ilmn
  Illumina multiplexing guide
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