High‐Throughput Multiplex Sequencing of miRNA

Francois Vigneault1, Dmitry Ter‐Ovanesyan2, Shahar Alon3, Seda Eminaga4, Danos C. Christodoulou4, J. G. Seidman4, Eli Eisenberg3, George M. Church5

1 Ragon Institute of MGH, MIT, and Harvard, Charlestown, Massachusetts, 2 Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, 3 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel, 4 Department of Genetics, Harvard Medical School, Boston, Massachusetts, 5 Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 11.12
DOI:  10.1002/0471142905.hg1112s73
Online Posting Date:  April, 2012
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Abstract

Next‐generation sequencing offers many advantages over other methods of microRNA (miRNA) expression profiling, such as sample throughput and the capability to discover novel miRNAs. As the sequencing depth of current sequencing platforms exceeds what is necessary to quantify miRNAs, multiplexing several samples in one sequencing run offers a significant cost advantage. Although previous studies have achieved this goal by adding bar codes to miRNA libraries at the ligation step, this was recently shown to introduce significant bias into the miRNA expression data. This bias can be avoided, however, by bar coding the miRNA libraries at the PCR step instead. Here, we describe a user‐friendly PCR bar‐coding method of preparing multiplexed microRNA libraries for Illumina‐based sequencing. The method also prevents the production of adapter dimers and can be completed in one day. Curr. Protoc. Hum. Genet. 73:11.12.1‐11.12.10 © 2012 by John Wiley & Sons, Inc.

Keywords: miRNA; Illumina; sequencing; library; multiplex; bar code

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • RNase Zap (Ambion, cat. no. AM9780)
  • Nuclease‐free water (Ambion, cat. no. AM9937)
  • Starting RNA
  • 10× T4 RNA Ligase 2tr buffer (Enzymatics, cat. no. L6070L)
  • 3′rApp‐adapter (see Table 11.12.1)
  • 100% dimethyl sulfoxide (DMSO; Sigma, cat. no. D9170)
  • RNase inhibitor (Enzymatics, cat. no. Y9240L)
  • RT primer (see Table 11.12.1)
  • 5′ RNA adapter (see Table 11.12.1)
  • ATP (Enzymatics, cat. no. N207‐10‐L)
  • T4 RNA ligase 1 (Enzymatics, cat. no. L6050L)
  • Superscript III First‐Strand Synthesis System (Invitrogen, cat. no. 18080051) containing:
    • 5× First‐strand buffer
    • DTT
  • dNTPs (Enzymatics, cat. no. N2050L)
  • Phusion High‐Fidelity DNA Polymerase (NEB, M0530S) containing:
    • 5× HF buffer
  • BCmiRNA_PCR1 (see Table 11.12.1)
  • BCmiRNA_PCR2_BC (see Table 11.12.1)
  • AgencourtAMPure XP kit (Beckman Coulter Genomics, cat. no. A63880)
  • 70% (v/v) ethanol
  • 2% E‐Gel EX Gel (Invitrogen, cat. no. G4020‐02)
  • 25‐bp ladder (Invitrogen, cat. no. 10597‐011)
  • 100‐bp ladder (Invitrogen, cat. no. 15628‐019)
  • MinElute Gel Extraction kit (Qiagen, cat. no. 28604)
  • Agilent High Sensitivity DNA Kit (Agilent, 5067‐4626)
  • 200‐µl PCR tubes
  • Thermal cycler (for all incubations)
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Dynamag‐2 Magnet (Invitrogen, cat. no. 123‐21D)
  • Microcentrifuge
  • E‐Gel I‐Base Power System (Invitrogen, cat. no. G6400)
  • E‐Gel Safe Imager Real‐Time Transilluminator (Invitrogen, G6500)
  • Razor blades
  • Nanodrop Spectrophotometer 2000, optional
  • Agilent 2100 Bioanalyzer
    Table 1.2.1   Materials   List of Oligonucleotides a   List of Oligonucleotides

    Name Sequence (5′‐3′)
    BCmiRNA_3′rApp‐adapter /5rApp/ACGGG′CTAATATTTATCGGTGG/3SpC3/
    BCmiRNA_5′RNA‐adapter rUrCrCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrC
    BCmiRNA_RT primer GCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR1 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
    BCmiRNA_PCR2‐BC1 CAAGCAGAAGACGGCATACGAGATCGATGTGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC2 CAAGCAGAAGACGGCATACGAGATTTAGGCGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC3 CAAGCAGAAGACGGCATACGAGATTGACCAGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC4 CAAGCAGAAGACGGCATACGAGATACGGTGGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC5 CAAGCAGAAGACGGCATACGAGATGCCAATGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC6 CAAGCAGAAGACGGCATACGAGATCAGATCGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC7 CAAGCAGAAGACGGCATACGAGATACTTGAGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC8 CAAGCAGAAGACGGCATACGAGATGATCAGGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC9 CAAGCAGAAGACGGCATACGAGATTAGCTTGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC10 CAAGCAGAAGACGGCATACGAGATGGCTACGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC11 CAAGCAGAAGACGGCATACGAGATCTTGTAGCTCCACCGATAAATATTAGCCCGT
    BCmiRNA_PCR2‐BC12 CAAGCAGAAGACGGCATACGAGATATCACGGCTCCACCGATAAATATTAGCCCGT
    BC_Custom_Indexing ACGGGCTAATATTTATCGGTGGAGC (optional)

     aNotes: All oligonucleotides can be ordered through Integrated DNA Technologies (IDT; http://www.idtdna.com), with HPLC purification. The bar‐codes are designed to be read in a single pass read, but a custom indexing primer can also be used if desired. The adenylated adapter can be ordered from IDT, or if on a budget, made as previously described (Vigneault et al., ).
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Figures

Videos

Literature Cited

Literature Cited
   Alon, S., Vigneault, F., Eminaga, S., Christodoulou, D.C., Seidman, J.G., Church, G.M., and Eisenberg, E. 2011. Barcoding bias in high‐throughput multiplex sequencing of miRNA. Genome Res. 21:1506‐1511.
   Creighton, C.J., Reid, J.G., and Gunaratne, P.H. 2009. Expression profiling of microRNAs by deep sequencing. Brief. Bioinform. 10:490‐497.
   Hafner, M., Renwick, N., Brown, M., Mihailović, A., Holoch, D., Lin, C., Pena, J.T., Nusbaum, J.D., Morozov, P., Ludwig, J., Ojo, T., Luo, S., Schroth, G., and Tuschl, T. 2011. RNA‐ligase‐dependent biases in miRNA representation in deep‐sequenced small RNA cDNA libraries. RNA 17:1697‐1712.
   Kawano, M., Kawazu, C., Lizio, M., Kawaji, H., Carninci, P., Suzuki, H., and Hayashizaki, Y. 2010. Reduction of non‐insert sequence reads by dimer eliminator LNA oligonucleotide for small RNA deep sequencing. BioTechniques 49:751‐755.
   Kloosterman, W.P. and Plasterk, R.H.A. 2006. The diverse functions of microRNAs in animal development and disease. Dev. Cell 11:441‐450.
   Lau, N.C., Lim, L.P., Weinstein, E.G., and Bartel, D.P. 2001. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858‐862.
   Tarasov, V., Jung, P., Verdoodt, B., Lodygin, D., Epanchintsev, A., Menssen, A., Meister, G., and Hermeking, H. 2007. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR‐34a is a p53 target that induces apoptosis and G1‐arrest. Cell Cycle 6:1586‐1593.
   Thomas, M.F. and Ansel, K.M. 2010. Construction of small RNA cDNA libraries for deep sequencing. Methods Mol. Biol. 667:93‐111.
   Uziel, T., Karginov, F.V., Xie, S., Parker, J.S., Wang, Y.‐D., Gajjar, A., He, L., Ellison, D., Gilbertson, R.J., Hannon, G., and Roussel, M.F. 2009. The miR‐17∼92 cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma. Proc. Natl. Acad. Sci. U.S.A. 106:2812‐2817.
   Vigneault, F., Sismour, A.M., and Church, G.M. 2008. Efficient microRNA capture and bar‐coding via enzymatic oligonucleotide adenylation. Nat. Methods 5:777‐779.
   Zhu, J.Y., Pfuhl, T., Motsch, N., Barth, S., Nicholls, J., Grässer, F., and Meister, G. 2009. Identification of novel Epstein‐Barr virus microRNA genes from nasopharyngeal carcinomas. J. Virol. 83:3333‐3341.
   Zhu, Q.‐H., Spriggs, A., Matthew, L., Fan, L., Kennedy, G., Gubler, F., and Helliwell, C. 2008. A diverse set of microRNAs and microRNA‐like small RNAs in developing rice grains. Genome Res. 18:1456‐1465.
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