Development of Poly(A)-ClickSeq as a tool enabling simultaneous genome-wide poly(A)-site identification and differential expression analysis
Development of Poly(A)-ClickSeq as a tool enabling simultaneous genome-wide poly(A)-site identification and differential expression analysis
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DOI：
10.1016/j.ymeth.2019.01.002
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发表时间：
2019-02-15
期刊：
影响因子：
4.8
通讯作者：
Routh, Andrew
中科院分区：
文献类型：
--
作者：
Elrod, Nathan D.;Jaworski, Elizabeth A.;Routh, Andrew
使用RNA-seq作为测量基因差异表达的通用工具，基本上取代了微阵列的使用。尽管这种方法具有公认的技术优势，但由于每个样品所需的基础设施、专业知识和时间，RNA-seq文库制备仍然主要由核心设施而不是在实验室进行。我们最近描述了两种基于“点击化学”的文库构建方法，称为ClickSeq和Poly（A）-ClickSeq（PAC-seq），作为传统RNA-seq的替代方案，它们既具有成本效益，又依赖于大多数实验室容易获得的简单试剂。ClickSeq是随机引物，可以对任何（未片段化的）RNA模板进行测序，而PAC-seq则针对mRNA的poly（A）尾。在这里，我们进一步开发了PAC-seq作为一个平台，允许同时定位poly（A）位点和测量基因的差异表达。我们提供了一个详细的协议，适当的计算管道的描述，以及一个原理证明数据集来说明该技术。PAC-seq提供了优于其他3'端作图方案的独特优势，因为它不需要额外的纯化、选择或片段化步骤，从而允许直接从粗总细胞RNA制备样品。我们已经证明，PAC-seq能够准确和灵敏地计数差异基因表达分析的转录本，以及识别替代的poly（A）位点和确定poly（A）尾边界的精确核苷酸。
The use of RNA-seq as a generalized tool to measure the differential expression of genes has essentially replaced the use of the microarray. Despite the acknowledged technical advantages to this approach, RNA-seq library preparation remains mostly conducted by core facilities rather than in the laboratory due to the infrastructure, expertise and time required per sample. We have recently described two 'click-chemistry' based library construction methods termed ClickSeq and Poly(A)-ClickSeq (PAC-seq) as alternatives to conventional RNA-seq that are both cost effective and rely on straightforward reagents readily available to most labs. ClickSeq is random primed and can sequence any (unfragmented) RNA template, while PAC-seq is targeted to poly(A) tails of mRNAs. Here, we further develop PAC-seq as a platform that allows for simultaneous mapping of poly(A) sites and the measurement of differential expression of genes. We provide a detailed protocol, descriptions of appropriate computational pipelines, and a proof-of-principle dataset to illustrate the technique. PAC-seq offers a unique advantage over other 3' end mapping protocols in that it does not require additional purification, selection, or fragmentation steps allowing sample preparation directly from crude total cellular RNA. We have shown that PAC-seq is able to accurately and sensitively count transcripts for differential gene expression analysis, as well as identify alternative poly(A) sites and determine the precise nucleotides of the poly(A) tail boundaries.