Advertisement

DNA/RNA Hybrid Primer Mediated Poly(A) Tag Library Construction for Illumina Sequencing

  • Man Liu
  • Xiaohui Wu
  • Qingshun Quinn LiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1255)

Abstract

Alternation polyadenylation is widespread in eukaryotes, and has demonstrated roles in gene expression regulation. Owing to deep DNA sequencing technologies, global analyses of alternation polyadenylation and their functions have become possible. We present a method to generate poly(A) tags libraries for high-throughput sequencing (PAT-seq). This protocol targets the junction of the 3′-UTR and poly(A) tail of a transcript so it can be positively identified as a poly(A) site. Upon Zinc-mediated limited digestion of total RNA, RNA fragments with poly(A) tail are then isolated and 5′-end repaired. A DNA/RNA hybrid adaptor is ligated to the 5′ end as an anchor. Then the library is generated by reverse transcription with oligo(dT)-adapter followed by PCR amplification. Such a custom poly(A) tags library can be generated from any source poly(A) containing RNA and good for both single- or paired-end sequencing in any Illumina sequencing platforms. This new method has been applied to investigate mRNA polyadenylation in Arabidopsis.

Key words

Polyadenylation Illumina sequencing Arabidopsis PAT-seq DNA/RNA hybrid primer 

Notes

Acknowledgement

We thank other lab members for helpful discussion and testing of the protocol. This work was supported by US National Science Foundation (grant nos. IOS–0817829 and IOS-1353354 to QQL), and a grant from Ohio Plant Biotech Consortium.

References

  1. 1.
    Elkon R, Ugalde AP, Agami R (2013) Alternative cleavage and polyadenylation: extent, regulation and function. Nat Rev Genet 14(7):496–506PubMedCrossRefGoogle Scholar
  2. 2.
    Xing D, Li QQ (2011) Alternative polyadenylation and gene expression regulation in plants. Wiley Interdiscip Rev RNA 2(3):445–458PubMedCrossRefGoogle Scholar
  3. 3.
    Shi Y (2012) Alternative polyadenylation: new insights from global analyses. RNA 18(12):2105–2117PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Tian B, Hu J, Zhang H, Lutz CS (2005) A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res 33(1):201–212PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Shepard PJ et al (2011) Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq. RNA 17(4):761–772PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Wu X et al (2011) Genome-wide landscape of polyadenylation in Arabidopsis provides evidence for extensive alternative polyadenylation. Proc Natl Acad Sci U S A 108(30):12533–12538PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Loke JC et al (2005) Compilation of mRNA polyadenylation signals in Arabidopsis revealed a new signal element and potential secondary structures. Plant Physiol 138(3):1457–1468PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Ji Z, Lee JY, Pan Z, Jiang B, Tian B (2009) Progressive lengthening of 3′ untranslated regions of mRNAs by alternative polyadenylation during mouse embryonic development. Proc Natl Acad Sci U S A 106(17):7028–7033PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Jan CH, Friedman RC, Ruby JG, Bartel DP (2011) Formation, regulation and evolution of Caenorhabditis elegans 3′UTRs. Nature 469(7328):97–101PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of BiologyMiami UniversityOxfordUSA
  2. 2.Department of AutomationXiamen UniversityXiamenChina
  3. 3.Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, College of the Environment and EcologyXiamen UniversityXiamenChina

Personalised recommendations