High throughput characterizations of poly(A) site choice in plants

Liuyin Ma; Pratap Kumar Pati; Man Liu; Qingshun Q. Li; Arthur G. Hunt

doi:10.1016/j.ymeth.2013.06.037

High throughput characterizations of poly(A) site choice in plants

Liuyin Ma, Pratap Kumar Pati, Man Liu, Qingshun Q. Li, Arthur G. Hunt

Research output: Contribution to journal › Article › peer-review

35 Scopus citations

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.

Original language	English
Pages (from-to)	74-83
Number of pages	10
Journal	Methods
Volume	67
Issue number	1
DOIs	https://doi.org/10.1016/j.ymeth.2013.06.037
State	Published - May 1 2014

Bibliographical note

Funding Information:
This work was supported by US National Science Foundation (IOS-0817818 to AGH and QQL), US National Institutes of Health (1R15GM094732-01 A1 to QQL), a grant from Ohio Plant Biotech Consortium to QQL, the University of Kentucky Executive Vice President for Research, and the Miami University Office for the Advancement of Research and Scholarship. Liuyin Ma was a recipient of a scholarship from the China Scholarship Council, and Dr. Pati was supported by a Fulbright-Nehru Senior Research Fellowship. The authors thank Ms. Carol Von Lanken for excellent technical assistance and Dr. Bruce Downie for helpful suggestions with the manuscript.

Keywords

Alternative polyadenylation
Gene expression
High throughput sequencing

ASJC Scopus subject areas

Molecular Biology
General Biochemistry, Genetics and Molecular Biology

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10.1016/j.ymeth.2013.06.037

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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.",

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author = "Liuyin Ma and Pati, {Pratap Kumar} and Man Liu and Li, {Qingshun Q.} and Hunt, {Arthur G.}",

note = "Funding Information: This work was supported by US National Science Foundation (IOS-0817818 to AGH and QQL), US National Institutes of Health (1R15GM094732-01 A1 to QQL), a grant from Ohio Plant Biotech Consortium to QQL, the University of Kentucky Executive Vice President for Research, and the Miami University Office for the Advancement of Research and Scholarship. Liuyin Ma was a recipient of a scholarship from the China Scholarship Council, and Dr. Pati was supported by a Fulbright-Nehru Senior Research Fellowship. The authors thank Ms. Carol Von Lanken for excellent technical assistance and Dr. Bruce Downie for helpful suggestions with the manuscript. ",

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N1 - Funding Information: This work was supported by US National Science Foundation (IOS-0817818 to AGH and QQL), US National Institutes of Health (1R15GM094732-01 A1 to QQL), a grant from Ohio Plant Biotech Consortium to QQL, the University of Kentucky Executive Vice President for Research, and the Miami University Office for the Advancement of Research and Scholarship. Liuyin Ma was a recipient of a scholarship from the China Scholarship Council, and Dr. Pati was supported by a Fulbright-Nehru Senior Research Fellowship. The authors thank Ms. Carol Von Lanken for excellent technical assistance and Dr. Bruce Downie for helpful suggestions with the manuscript.

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N2 - 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.

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High throughput characterizations of poly(A) site choice in plants

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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