SHAPE–Seq: High‐Throughput RNA Structure Analysis

Stefanie A. Mortimer1, Cole Trapnell2, Sharon Aviran2, Lior Pachter3, Julius B. Lucks4

1 Department of Molecular and Cell Biology, University of California, Berkeley, California, 2 These authors contributed equally to this work., 3 Department of Mathematics, University of California, Berkeley, California, 4 School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120019
Online Posting Date:  December, 2012
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Abstract

Knowledge of RNA structure is critical to understanding both the important functional roles of RNA in biology and the engineering of RNA to control biological systems. This article contains a protocol for selective 2′‐hydroxyl acylation analyzed by primer extension and sequencing (SHAPE‐Seq) that, through a combination of structure‐dependent chemical probing and next‐generation sequencing technologies, achieves structural characterization of hundreds of RNAs in a single experiment. This protocol is applicable in a variety of conditions, and represents an important tool for understanding RNA biology. The protocol includes methods for the design and synthesis of RNA mixtures for study, and the construction and analysis of structure‐dependent sequencing libraries that reveal structural information of the RNAs in the mixtures. The methods are generally applicable to studying RNA structure and interactions in vitro in a variety of conditions, and allows for the rapid characterization of RNA structures in a high‐throughput manner. Curr. Protoc. Chem. Biol. 4:275‐297 © 2012 by John Wiley & Sons, Inc.

Keywords: RNA structure; next‐generation sequencing; chemical probing; high throughput

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of a Barcoded RNA Library through PCR and In Vitro Transcription
  • Basic Protocol 2: Preparation of a Structure‐Specific cDNA Library for Paired‐End Sequencing
  • Basic Protocol 3: Read Alignment and Calculation of Shape Reactivities
  • Support Protocol 1: Synthesis of 1‐Methyl‐7‐Nitroisatoic Anhydride (1M7)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of a Barcoded RNA Library through PCR and In Vitro Transcription

  Materials
  • Forward and reverse PCR primers at 100 µM (custom‐synthesized by Integrated DNA Technologies, https://www.idtdna.com/)
  • PCR buffer (without Mg2+; Invitrogen, cat. no. 18067‐017)
  • 1 M MgCl 2
  • 10 mM dNTP mixture (dATP, dCTP, dGTP, dTTP; New England Biolabs, cat. no. N0447L)
  • Template DNA for PCR: dsDNA containing RNA coding sequence of interest (circular or linear)
  • Taq DNA polymerase (Invitrogen, cat. no. 10342‐020)
  • 4 M NaCl
  • 100% ethanol
  • 1 M Tris⋅Cl, pH 8.0
  • 1 M DTT (Invitrogen, cat. no P2325)
  • Spermidine (Sigma, cat. no. 85558)
  • Triton X‐100 (Sigma, cat. no. T8787)
  • NTPs: ATP, Sigma, cat no. A7699‐1G), UTP (Sigma, cat. no. U6625‐1G), CTP (Sigma, cat. no. C1506‐1G), GTP (Sigma, cat. no. G8877‐1G)
  • RNase Inhibitor (Promega, cat. no. N2615)
  • T7 RNA polymerase (Ambion, cat. no. 18033‐019)
  • 3 M sodium acetate, pH 5.2
  • Stop dye (see recipe)
  • Nuclease‐free water
  • TE buffer (see recipe)
  • Additional reagents and equipment for PCR (Kramer and Coen, ), agarose gel electrophoresis (Voytas, ), and purification of RNA by denaturing polyacrylamide gel electrophoresis (Ellington and Pollard, )

Basic Protocol 2: Preparation of a Structure‐Specific cDNA Library for Paired‐End Sequencing

  Materials
  • RNA ( protocol 1)
  • Nuclease‐free water
  • 3.3× folding buffer (see recipe)
  • SHAPE reagent (one of the following):
    • 65 mM 1‐methyl‐7‐nitroisatoic anhydride (1M7; see recipe)
    • 65 mM NMIA (see recipe)
    • 400 mM benzoyl cyanide (BzCN; see recipe)
  • 3 M sodium acetate, pH 5.2
  • 20 mg/ml glycogen (Invitrogen, cat. no 10814‐010)
  • 100% ethanol
  • 3 µM Primer A and B (custom‐synthesized by Integrated DNA Technologies, https://www.idtdna.com/; see Fig. )
  • Enzyme master mix (see recipe)
  • SuperScript III reverse transcriptase (Invitrogen, cat. no. 18080‐044)
  • 4 M NaOH
  • 10× CircLigase ligation buffer (see recipe)
  • 50 mM MnCl 2
  • 1 mM ATP
  • A_Adapter_b with 5′ phosphate and 3′ C3 (propyl) modification at 100 µM (custom‐synthesized by Integrated DNA Technologies, https://www.idtdna.com/, PAGE‐purified; see Fig. )
  • CircLigase ssDNA ligase (Epicentre Biotechnologies, cat. no. CL4111K)
  • Agencourt XP purification beads (Beckman Coulter, cat. no. A63880)
  • 70% ethanol
  • TE buffer (see recipe)
  • 5× Phusion high‐fidelity buffer (NEB, cat. no M0530S)
  • 10 mM dNTP mixture (dATP, dCTP, dGTP, dTTP; New England Biolabs, cat. no. N0447L)
  • 100 µM Primers PE_F and PE_R at 100 µM (IDT) (Fig. )
  • Phusion high fidelity DNA polymerase (NEB, cat. no M0530S)
  • Thermal cycler with heated lid
  • Agencourt XP magnetic stand (Beckman Coulter, cat. no. A29182)
  • Agilent Bioanalyzer high‐sensitivity DNA kit (Agilent, cat. no 5067‐4626)

Basic Protocol 3: Read Alignment and Calculation of Shape Reactivities

  Materials
  • Python: Python is a common programming language found on many platforms by default. Python installation instructions can be found at http://python.org for a variety of systems.
  • Bowtie: The bowtie short read aligner and installation instructions can be downloaded from http://bowtie‐bio.sourceforge.net/index.shtml.
  • The Boost Libraries: Boost is a collection of open‐source programming utilities that provide advanced numerical capabilities relied upon by the Spats SHAPE‐Seq pipeline. Boost can be downloaded by following instructions at http://www.boost.org.
  • Spats package: The Spats package can be downloaded from http://spats.sourceforge.net/. Complete installation instructions for Spats can be found in the documentation on the Web site.

Support Protocol 1: Synthesis of 1‐Methyl‐7‐Nitroisatoic Anhydride (1M7)

  Materials
  • 4‐Nitro‐isatoic anhydride (AstaTech, cat. no. 69441; http://www.astatechinc.com/)
  • Sodium hydride (60% dispersion in mineral oil; Sigma‐Aldrich, cat. no. 452912‐100G)
  • Iodomethane (Sigma, cat. no. 289566)
  • N,N‐dimethylformamide (DMF) (anhydrous, Sigma, cat. no. 227056)
  • 1 N hydrochloric acid solution (1 N Certified; Fisher, cat. no. 7647‐01‐0), cold
  • Ether
  • Flame or oven‐dried round bottom flasks (2) fitted with magnetic stir bar
  • Magnetic stir plate
  • Watch glass
  • Additional reagents and equipment for mass spectrometry and NMR
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Figures

Videos

Literature Cited

   Amaral, P.P., Dinger, M.E., Mercer, T.R., and Mattick, J.S. 2008. The eukaryotic genome as an RNA machine. Science 319:1787‐1789.
   Aviran, S., Lucks, J.B., and Pachter, L. 2011a. RNA structure characterization from chemical mapping experiments. Proc. 49th Allerton Conf. on Communication, Control and Computing 1743‐1750. IEEE Press, New York.
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   Butcher, S.E. and Pyle, A.M. 2011. The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks. Acc. Chem. Res. 44:1302‐1311.
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   Kladwang, W., Vanlang, C.C., Cordero, P., and Das, R. 2011. Understanding the errors of SHAPE‐directed RNA structure modeling. Biochemistry 50:8049‐8056.
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   Lucks, J.B., Mortimer, S.A., Trapnell, C., Luo, S., Aviran, S., Schroth, G.P., Pachter, L., Doudna, J.A., and Arkin, A.P. 2011. Multiplexed RNA structure characterization with selective 2′‐hydroxyl acylation analyzed by primer extension sequencing (SHAPE‐Seq). Proc. Natl. Acad. Sci. U.S.A. 108:11063‐11068.
   Merino, E.J., Wilkinson, K.A., Coughlan, J.L., and Weeks, K.M. 2005. RNA structure analysis at single nucleotide resolution by selective 2′‐hydroxyl acylation and primer extension (SHAPE). J. Am. Chem. Soc. 127:4223‐4231.
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   Mortimer, S.A. and Weeks, K.M. 2007. A fast‐acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J. Am. Chem. Soc. 129:4144‐4145.
   Mortimer, S.A. and Weeks, K.M. 2008. Time‐resolved RNA SHAPE chemistry. J. Am. Chem. Soc. 130:16178‐16180.
   Mortimer, S.A. and Weeks, K.M. 2009. Time‐resolved RNA SHAPE chemistry: Quantitative RNA structure analysis in one‐second snapshots and at single‐nucleotide resolution. Nat. Protoc. 4:1413‐1421.
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   Steen, K.‐A., Arun, M., and Weeks, K.M. 2010. Selective 2′‐hydroxyl acylation analyzed by protection from exoribonuclease. J. Am. Chem. Soc. 132:9940‐9943.
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Internet Resources
   http://spats.sourceforge.net/
  Download site for Spats, a program for the analysis of sequencing information to determine SHAPE reactivities in a SHAPE‐Seq experiment.
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Supplementary Material

cbv4n4.zip

The above zip file contains:

1. Example output for the single WT RNaseP sequence. (Sample_output_RNaseP_WT.out)

2. Example output for a mixed library. (Sample_output_mixed_library.out)

3. A sample FASTA file containing a target RNA sequence for each RNA in the mixture in the experiment. (target_pool.fa)

Note: these files should be opened with WordPad.