Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA.
Methods. 2019 Feb 15;155:20-29. doi: 10.1016/j.ymeth.2019.01.002. Epub 2019 Jan 6.
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.
RNA-seq 作为一种通用的工具，用于测量基因的差异表达，已经基本取代了微阵列。尽管这种方法在技术上有公认的优势，但由于每个样本所需的基础设施、专业知识和时间，RNA-seq 文库制备仍然主要由核心设施完成，而不是在实验室中进行。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），它们是替代传统 RNA-seq 的方法，既具有成本效益，又依赖于大多数实验室都容易获得的简单试剂。ClickSeq 是随机引物的，可以对任何（未片段化）RNA 模板进行测序，而 PAC-seq 则靶向 mRNAs 的 poly(A)尾巴。在这里，我们进一步将 PAC-seq 开发为一个平台，允许同时对 poly(A)位点进行映射和基因差异表达的测量。我们提供了详细的方案、适当的计算管道描述以及一个原理验证数据集，以说明该技术。与其他 3'端映射协议相比，PAC-seq 具有一个独特的优势，即它不需要额外的纯化、选择或片段化步骤，允许直接从粗总细胞 RNA 中进行样品制备。我们已经表明，PAC-seq 能够准确和敏感地计算差异基因表达分析的转录本，以及识别替代的 poly(A)位点并确定 poly(A)尾巴边界的精确核苷酸。
