Ma Liuyin, Pati Pratap Kumar, Liu Man, Li Qingshun Q, Hunt Arthur G
Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA.
Department of Horticulture, University of Kentucky, Lexington, KY, USA; Department of Biotechnology, Guru Nanak Dev University, Amritsar, India.
Methods. 2014 May 1;67(1):74-83. doi: 10.1016/j.ymeth.2013.06.037. Epub 2013 Jul 11.
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.
真核生物中mRNA的聚腺苷酸化是一个重要的生物学过程。近年来，由于新一代DNA测序技术的出现，mRNA聚腺苷酸化领域取得了重大进展。高通量测序能力使得大量聚腺苷酸化位点得以直接通过实验确定，对这些位点的分析揭示了真核生物中基因表达调控的巨大潜力。这些数据集是使用专门针对3'-UTR与聚（A）尾连接处的测序方法生成的。在此，我们展示了用于分析植物中可变聚腺苷酸化的该方案的三种变体。虽然所有这些方法都使用寡聚dT作为3'-末端的锚定物，但它们在为5'-末端生成锚定物的方式上有所不同，以便产生适合Illumina有效测序的PCR产物；使用不同方法添加5'接头扩展了这些方法的可能用途。这些方法通用性强、可重复，可用于基因表达分析以及聚（A）位点选择的全局测定。
