Liu Man, Wu Xiaohui, Li Qingshun Quinn
Department of Biology, Miami University, Oxford, OH, 45056, USA.
Methods Mol Biol. 2015;1255:175-84. doi: 10.1007/978-1-4939-2175-1_15.
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.
可变聚腺苷酸化在真核生物中广泛存在，并已证明在基因表达调控中发挥作用。由于深度DNA测序技术的出现，对可变聚腺苷酸化及其功能进行全局分析成为可能。我们提出了一种用于高通量测序的生成聚（A）标签文库的方法（PAT-seq）。该方案针对转录本的3'-UTR和聚（A）尾的连接处，因此可以明确鉴定为聚（A）位点。在用锌介导的总RNA有限消化后，分离出带有聚（A）尾的RNA片段并进行5'端修复。将DNA/RNA杂交接头连接到5'端作为锚定。然后通过用寡聚（dT）-接头进行逆转录，随后进行PCR扩增来生成文库。这样的定制聚（A）标签文库可以从任何含有聚（A）的RNA来源生成，适用于任何Illumina测序平台的单端或双端测序。这种新方法已应用于研究拟南芥中的mRNA聚腺苷酸化。
