High throughput characterizations of poly(A) site choice in plants.
High throughput characterizations of poly(A) site choice in plants.
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DOI：
10.1016/j.ymeth.2013.06.037
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发表时间：
2014-05-01
期刊：
影响因子：
4.8
通讯作者：
Hunt, Arthur G.
中科院分区：
文献类型：
--
作者：
Ma, Liuyin;Pati, Pratap Kumar;Liu, Man;Li, Qingshun Q.;Hunt, Arthur G.
真核生物中mRNA的多聚腺苷酸化是一个重要的生物学过程。近年来，由于下一代DNA测序技术的出现，mRNA多腺苷酸化领域取得了重大进展。高通量测序能力已经导致大量多聚腺苷酸化位点的直接实验测定，其分析揭示了真核生物基因表达调控的巨大潜力。这些集合是使用专门的测序方法生成的，这些方法针对 3'-UTR 和 Poly(A) 尾的连接处。在这里，我们提出了这种方案的三种变体，该方案已用于分析植物中的替代聚腺苷酸化。虽然所有这些方法都使用oligo(dT)作为3'端的锚定点，但它们在生成5'端锚定点的方法上有所不同，以产生适合有效Illumina测序的PCR产物；使用不同的方法附加 5' 适配器扩展了这些方法的可能用途。这些方法用途广泛、重现性好，可用于基因表达分析以及 Poly(A) 位点选择的全局测定。
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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