Strand‐Specific Transcriptome Sequencing Using SMART Technology

Magnolia Bostick1, Nathalie Bolduc1, Alisa Lehman1, Andrew Farmer1

1 Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.27
DOI:  10.1002/cpmb.22
Online Posting Date:  October, 2016
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Abstract

Next‐generation sequencing is empowering a deeper understanding of biology by enabling RNA expression analysis over the entire transcriptome with high sensitivity and a wide dynamic range. One powerful application within this field is stranded RNA sequencing (RNA‐seq), which is necessary to distinguish overlapping genes and to conduct comprehensive annotation and quantification of long non‐coding RNAs. Commonly used methods for generating strand‐specific RNA‐seq libraries are often complicated by protocols that require several rounds of enzymatic treatments and clean‐up steps, making them time‐intensive, insensitive, and unsuitable for processing several samples simultaneously. An additional challenge in the generation of RNA‐seq libraries from total RNA involves the high amount of ribosomal RNA (rRNA) in the starting material. This unit presents streamlined workflows for generating strand‐specific RNA‐seq libraries from 10 ng to 1 µg total RNA, representing a minimum of 1000 cells, in less than 7 hr with minimal carryover rRNA. These methods allow scientists to evaluate the expression of all transcripts, including non‐polyadenylated long non‐coding RNAs, even in limited biological samples. Combination of the RNase H–based RiboGone rRNA removal system and SMARTer Stranded RNA‐seq technology enables depletion of over 95% of rRNA from mammalian samples, and direct production of Illumina‐ready libraries that maintain strand‐of‐origin information. An alternate method for low input of highly degraded samples is also presented. © 2016 by John Wiley & Sons, Inc.

Keywords: stranded RNA‐seq; rRNA removal; non‐coding RNA; degraded RNA

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: rRNA Removal For Low‐Input Mammalian Total RNA
  • Basic Protocol 2: Strand‐Specific cDNA Synthesis and Library Preparation
  • Alternate Protocol 1: Complete RNA‐SEQ Library Preparation from Mammalian Total RNA
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: rRNA Removal For Low‐Input Mammalian Total RNA

  Materials
  • Total RNA
  • RiboGone Mammalian Kit (Clontech, cat. no. 634846 or 634847) containing:
    • 5× RiboGone Hyb Buffer
    • Nuclease‐free water
    • RNase H
    • 10× RNase H Buffer
    • 40 U/µl RNase Inhibitor
    • DNase I (5 U/µl)
    • RiboGone Purification Buffer
  • Agencourt AMPure XP PCR Purification Kit (Beckman Coulter, cat. no. A63880 or A63881)
  • 80% (v/v) ethanol, freshly prepared
  • 0.2‐ml nuclease‐free thin‐wall 8‐strip PCR tubes (GeneMate, cat. no. T‐3035‐1; or USA Scientific, cat. no. 1402‐4700)
  • Thermal cycler
  • Magnetic separation device

Basic Protocol 2: Strand‐Specific cDNA Synthesis and Library Preparation

  Materials
  • rRNA‐depleted total RNA (see protocol 1)
  • SMARTer Stranded RNA‐Seq Kit (Clontech, cat. no. 634836‐634839, 634861) or SMARTer Stranded RNA‐Seq Kit HT (Clontech, cat. no. 634862) containing:
    • 1 µg/µl Control Mouse Liver Total RNA
    • 12 µM SMARTer Stranded N6 Primer
    • 5× First‐Strand Buffer, RNase‐free
    • Nuclease‐free water
    • 100 mM dithiothreitol (DTT)
    • 40 U/μl RNase Inhibitor
    • 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, and dTTP)
    • 12 μM SMARTer Stranded Oligonucleotide
    • 100 U/µl SMARTScribe Reverse Transcriptase
    • 2× SeqAmp PCR Buffer
    • Illumina Indexing Primer Set or Indexing Primer Set HT
    • SeqAmp DNA Polymerase
    • Stranded Elution Buffer
  • Agencourt AMPure XP PCR Purification Kit (Beckman Coulter, cat. no. A63880 or A63881)
  • 80% (v/v) ethanol, freshly prepared
  • High‐Sensitivity DNA Kit for Bioanalyzer (Agilent, cat. no. 5067‐4626)
  • 0.2‐ml nuclease‐free thin‐wall 8‐strip PCR tubes (GeneMate, cat. no. T‐3035‐1; or USA Scientific, cat. no. 1402‐4700)
  • Hot‐lid thermal cycler
  • Magnetic separation device
  • Agilent 2100 Bioanalyzer

Alternate Protocol 1: Complete RNA‐SEQ Library Preparation from Mammalian Total RNA

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • SMARTer Stranded Total RNA Sample Prep Kit ‐ HI Mammalian (Clontech, cat. no. 634873‐634878) containing:
    • 1 μg/μl Control Mouse Liver Total RNA
    • 10× Total RNA Hyb Buffer
    • Nuclease‐free water
    • RNase H
    • 10× RNase H Buffer
    • 40 U/μl RNase Inhibitor
    • 5 U/μl DNase I
    • 10× Total RNA First‐Strand Buffer
    • 12 μM SMARTer Stranded Oligonucleotide
    • 200 U/μl PrimeScript Reverse Transcriptase
    • 2× SeqAmp PCR Buffer
    • Indexing Primer Set HT for Illumina (12, 48 A, or 96)
    • SeqAmp DNA Polymerase
    • Stranded Elution Buffer
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Figures

Videos

Literature Cited

Literature Cited
  Adiconis, X., Borges‐Rivera, D., Satija, R., DeLuca, D.S., Busby, M.A., Berlin, A.M., Sivachenko, A., Thompson, D.A., Wysoker, A., Fennell, T., Gnirke, A., Pochet, N., Regev, A., and Levin, J.Z. 2013. Comprehensive comparative analysis of RNA sequencing methods for degraded or low input samples. Nat. Methods 10:623‐629. doi: 10.1038/nmeth.2483.
  Armour, C.D., Castle, J.C., Chen, R., Babak, T., Loerch, P., Jackson, S., Shah, J.K., Dey, J., Rohl, C.A., Johnson, J.M., and Raymond, C.K. 2009. Digital transcriptome profiling using selective hexamer priming for cDNA synthesis. Nat. Methods 6:647‐649. doi: 10.1038/nmeth.1360.
  Chenchik, A., Zhu, Y., Diatchenko, L., Li, R., Hill, J., and Siebert, P. 1998. Generation and use of high‐quality cDNA from small amounts of total RNA by SMART PCR. In RT‐PCR Methods for Gene Cloning and Analysis (P. Siebert and J. Larrick, eds.) pp. 305‐319. BioTechniques Books, Natick, Mass.
  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. doi: 10.1038/nature11233.
  Hangauer, M.J., Vaughn, I.W., and McManus, M.T. 2013. Pervasive transcription of the human genome produces thousands of previously unidentified long intergenic noncoding RNAs. PLoS Genetics 9:e1003569. doi: 10.1371/journal.pgen.1003569.
  Hindorff, L.A., Sethupathy, P., Junkins, H.A., Ramos, E.M., Mehta, J.P., Collins, F.S., and Manolio, T.A. 2009. Potential etiologic and functional implications of genome‐wide association loci for human diseases and traits. Proc. Natl. Acad. Sci. U.S.A. 106:9362‐9367. doi: 10.1073/pnas.0903103106.
  Kornienko, A.E., Guenzl, P.M., Barlow, D.P., and Pauler, F.M. 2013. Gene regulation by the act of long non‐coding RNA transcription. BMC Biol. 11:59. doi: 10.1186/1741‐7007‐11‐59.
  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. doi: 10.1038/nmeth.1491.
  Mattick, J.S. and Makunin, I.V. 2006. Non‐coding RNA. Hum. Mol. Genet. 15:R17‐29. doi: 10.1093/hmg/ddl046.
  Mills, J.D., Kawahara, Y., and Janitz, M. 2013. Strand‐specific RNA‐seq provides greater resolution of transcriptome profiling. Curr. Genomics 14:173‐181. doi: 10.2174/1389202911314030003.
  Morlan, J.D., Qu, K., and Sinicropi, D.V. 2012. Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue. PLoS One 7:e42882. doi: 10.1371/journal.pone.0042882.
  Parkhomchuk, D., Borodina, T., Amstislavskiy, V., Banaru, M., Hallen, L., Krobitsch, S., Lehrach, H., and Soldatov, A. 2009. Transcriptome analysis by strand‐specific sequencing of complementary DNA. Nucleic Acids Res. 37:e123. doi: 10.1093/nar/gkp596.
  Wapinski, O. and Chang H.Y. 2011. Long noncoding RNAs and human disease. Trends Cell Biol. 21:354‐361. doi: 10.1016/j.tcb.2011.04.001.
  Yi, H., Cho, Y.J., Won, S., Lee, J.E., Yu, H.J., Kim, S., Schroth, G.P., Luo, S., and Chun, J. 2011. Duplex‐specific nuclease efficiently removes rRNA for prokaryotic RNA‐seq. Nucleic Acids Res. 39:e140. doi: 10.1093/nar/gkr617.
  Zhulidov, P.A., Bogdanova, E.A., Shcheglov, A.S., Vagner, L.L., Khaspekov, G.L., Kozhemyako, V.B., Matz, M.V., Meleshkevitch, E., Moroz, L.L., Lukyanov, S.A., and Shagin, D.A. 2004. Simple cDNA normalization using kamchatka crab duplex‐specific nuclease. Nucleic Acids Res. 32:e37. doi: 10.1093/nar/gnh031.
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