title: High throughput characterizations of poly(A) site choice in plants.
abstract: The polyadenylation of mRNA in eukaryotes is an important biological process. In recent years, significant progress has been made in the field of mRNA polyadenylation owing to the advent of the next generation DNA sequencing technologies. The high-throughput sequencing capabilities have resulted in the direct experimental determinations of large numbers of polyadenylation sites, analysis of which has revealed a vast potential for the regulation of gene expression in eukaryotes. These collections have been generated using specialized sequencing methods that are targeted to the junction of 3'-UTR and the poly(A) tail. Here we present three variations of such a protocol that has been used for the analysis of alternative polyadenylation in plants. While all these methods use oligo-dT as an anchor to the 3'-end, they differ in the means of generating an anchor for the 5'-end in order to produce PCR products suitable for effective Illumina sequencing; the use of different methods to append 5' adapters expands the possible utility of these approaches. These methods are versatile, reproducible, and may be used for gene expression analysis as well as global determinations of poly(A) site choice.
authors.affiliation: Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA.
authors.forename: Liuyin
authors.initials: L
authors.lastname: Ma
authors.affiliation: Department of Horticulture, University of Kentucky, Lexington, KY, USA; Department of Biotechnology, Guru Nanak Dev University, Amritsar, India.
authors.forename: Pratap Kumar
authors.initials: PK
authors.lastname: Pati
authors.affiliation: Department of Botany, Miami University, Oxford, OH, USA.
authors.forename: Man
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Botany, Miami University, Oxford, OH, USA; Key Laboratory of Ministry of Education for Coastal Wetland Ecosystem, College of the Environment and Ecology, Xiamen University, Fujian, China. Electronic address: liq@miamioh.edu.
authors.forename: Qingshun Q
authors.initials: QQ
authors.lastname: Li
authors.affiliation: Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA. Electronic address: aghunt00@uky.edu.
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Hunt
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D026723:Polyadenylation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D018749:RNA, Plant
keywords: Alternative polyadenylation; Gene expression; High throughput sequencing