A Rapid, Simple, and Inexpensive Method for the Preparation of Strand-Specific RNA-Seq Libraries

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1255)

Abstract

High-throughput sequencing of short cDNA tags, or RNA-Seq, has become a staple of genome-wide gene expression studies in plants. RNA-Seq libraries necessarily contain tags that correspond to the mRNA-poly(A) junction, or polyadenylation site, and thus may be mined for data that can help study alternative polyadenylation. This report presents a simple, rapid, and inexpensive method for preparing strand-specific RNA-Seq libraries from varying quantities of total RNA.

Key words

Alternative polyadenylation High-throughput sequencing Gene expression Strand-specific RNA-Seq 

Notes

Acknowledgements

This work was supported by the National Science Foundation (awards IOS-0817818 and MCB-1243849). The author thanks the staff of AGTC for many helpful discussions and for much patience, and Carol Von Lanken for excellent technical and administrative assistance.

References

  1. 1.
    Hunt AG (2008) Messenger RNA 3′ end formation in plants. Curr Top Microbiol Immunol 326:151–177PubMedGoogle Scholar
  2. 2.
    Tan X, Meyers BC, Kozik A, West MA, Morgante M, St Clair DA, Bent AF, Michelmore RW (2007) Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis. BMC Plant Biol 7:56. doi: 10.1186/1471-2229-7-56 PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Rataj K, Simpson GG (2014) Message ends: RNA 3′ processing and flowering time control. J Exp Bot 65(2):353–363. doi: 10.1093/jxb/ert439 PubMedCrossRefGoogle Scholar
  4. 4.
    Ma L, Pati PK, Liu M, Li QQ, Hunt AG (2013) High throughput characterizations of poly(A) site choice in plants. Methods. doi: 10.1016/j.ymeth.2013.06.037 Google Scholar
  5. 5.
    Thomas PE, Wu X, Liu M, Gaffney B, Ji G, Li QQ, Hunt AG (2012) Genome-wide control of polyadenylation site choice by CPSF30 in Arabidopsis. Plant Cell 24(11):4376–4388. doi: 10.1105/tpc.112.096107 PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Wu X, Liu M, Downie B, Liang C, Ji G, Li QQ, Hunt AG (2011) Genome-wide landscape of polyadenylation in Arabidopsis provides evidence for extensive alternative polyadenylation. Proc Natl Acad Sci U S A 108(30):12533–12538. doi: 10.1073/pnas.1019732108 PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Duc C, Sherstnev A, Cole C, Barton GJ, Simpson GG (2013) Transcription termination and chimeric RNA formation controlled by Arabidopsis thaliana FPA. PLoS Genet 9(10):e1003867. doi: 10.1371/journal.pgen.1003867 PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Lyons R, Iwase A, Gansewig T, Sherstnev A, Duc C, Barton GJ, Hanada K, Higuchi-Takeuchi M, Matsui M, Sugimoto K, Kazan K, Simpson GG, Shirasu K (2013) The RNA-binding protein FPA regulates flg22-triggered defense responses and transcription factor activity by alternative polyadenylation. Sci Rep 3:2866. doi: 10.1038/srep02866 PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Sherstnev A, Duc C, Cole C, Zacharaki V, Hornyik C, Ozsolak F, Milos PM, Barton GJ, Simpson GG (2012) Direct sequencing of Arabidopsis thaliana RNA reveals patterns of cleavage and polyadenylation. Nat Struct Mol Biol 19(8):845–852. doi: 10.1038/nsmb.2345 PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Janbon G, Ormerod KL, Paulet D, Byrnes EJ 3rd, Yadav V, Chatterjee G, Mullapudi N, Hon CC, Billmyre RB, Brunel F, Bahn YS, Chen W, Chen Y, Chow EW, Coppee JY, Floyd-Averette A, Gaillardin C, Gerik KJ, Goldberg J, Gonzalez-Hilarion S, Gujja S, Hamlin JL, Hsueh YP, Ianiri G, Jones S, Kodira CD, Kozubowski L, Lam W, Marra M, Mesner LD, Mieczkowski PA, Moyrand F, Nielsen K, Proux C, Rossignol T, Schein JE, Sun S, Wollschlaeger C, Wood IA, Zeng Q, Neuveglise C, Newlon CS, Perfect JR, Lodge JK, Idnurm A, Stajich JE, Kronstad JW, Sanyal K, Heitman J, Fraser JA, Cuomo CA, Dietrich FS (2014) Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation. PLoS Genet 10(4):e1004261. doi: 10.1371/journal.pgen.1004261 PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Schlackow M, Marguerat S, Proudfoot NJ, Bahler J, Erban R, Gullerova M (2013) Genome-wide analysis of poly(A) site selection in Schizosaccharomyces pombe. RNA 19(12):1617–1631. doi: 10.1261/rna.040675.113 PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Zhao Z, Wu X, Kumar PK, Dong M, Ji G, Li QQ, Liang C (2014) Bioinformatics analysis of alternative polyadenylation in green alga Chlamydomonas reinhardtii using transcriptome sequences from three different sequencing platforms. G3 (Bethesda) 4(5):871–883. doi: 10.1534/g3.114.010249 CrossRefGoogle Scholar
  13. 13.
    Jimenez-Gomez JM (2011) Next generation quantitative genetics in plants. Front Plant Sci 2:77. doi: 10.3389/fpls.2011.00077 PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    O'Rourke JA, Bolon YT, Bucciarelli B, Vance CP (2014) Legume genomics: understanding biology through DNA and RNA sequencing. Ann Bot 113(7):1107–1120. doi: 10.1093/aob/mcu072 PubMedCrossRefGoogle Scholar
  15. 15.
    Strickler SR, Bombarely A, Mueller LA (2012) Designing a transcriptome next-generation sequencing project for a nonmodel plant species. Am J Bot 99(2):257–266. doi: 10.3732/ajb.1100292 PubMedCrossRefGoogle Scholar
  16. 16.
    Ramskold D, Luo S, Wang YC, Li R, Deng Q, Faridani OR, Daniels GA, Khrebtukova I, Loring JF, Laurent LC, Schroth GP, Sandberg R (2012) Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol 30(8):777–782. doi: 10.1038/nbt.2282 PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD (2001) Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30(4):892–897PubMedGoogle Scholar
  18. 18.
    Buonaccorsi V, Peterson M, Lamendella G, Newman J, Trun N, Tobin T, Aguilar A, Hunt A, Praul C, Grove D, Roney J, Roberts W (2014) Vision and change through the genome consortium for active teaching using next-generation sequencing (GCAT-SEEK). CBE Life Sci Educ 13(1):1–2. doi: 10.1187/cbe.13-10-0195 PubMedCentralPubMedGoogle Scholar
  19. 19.
    Picelli S, Bjorklund AK, Faridani OR, Sagasser S, Winberg G, Sandberg R (2013) Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10(11):1096–1098. doi: 10.1038/nmeth.2639 PubMedCrossRefGoogle Scholar
  20. 20.
    Picelli S, Faridani OR, Bjorklund AK, Winberg G, Sagasser S, Sandberg R (2014) Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9(1):171–181. doi: 10.1038/nprot.2014.006 PubMedCrossRefGoogle Scholar
  21. 21.
    Pinto FL, Lindblad P (2010) A guide for in-house design of template-switch-based 5′ rapid amplification of cDNA ends systems. Anal Biochem 397(2):227–232. doi: 10.1016/j.ab.2009.10.022 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Plant and Soil SciencesUniversity of KentuckyLexingtonUSA

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