Cell type-specific transcriptome profiling in C. elegans using the Translating Ribosome Affinity Purification technique
Cell type-specific transcriptome profiling in C. elegans using the Translating Ribosome Affinity Purification technique
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DOI：
10.1016/j.ymeth.2017.06.023
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发表时间：
2017-08-15
期刊：
影响因子：
4.8
通讯作者：
Calarco, John A.
中科院分区：
文献类型：
--
作者：
Gracida, Xicotencatl;Calarco, John A.
器官和特定的细胞类型在多细胞生物中执行专门的功能，在很大程度上通过定制的基因表达特征。因此，分析特定细胞和组织类型的转录组仍然是理解细胞如何特化的重要工具。检测基因表达差异的方法学途径已经利用来自整个动物、解剖组织和最近的单细胞的样品。尽管取得了这些进展，但在大多数实验室中仍然存在挑战和需求，以实施侵入性较小但功能强大的细胞类型特异性转录组分析方法。在这里，我们描述了使用翻译核糖体亲和纯化（TRAP）的方法为C。elegans在翻译mRNA水平上检测细胞类型特异性基因表达模式。在TRAP中，核糖体蛋白与标签（GFP）融合，并在细胞类型特异性启动子下表达，以在体内标记遗传定义的细胞类型。表达标签的动物的裂解物的亲和纯化富集靶组织的核糖体相关mRNA。纯化的mRNA用于制备cDNA文库，进行高通量测序以获得来自靶细胞类型的转录物的全基因组谱。暴露C的容易性。通过将TRAP与可利用的细胞类型特异性启动子结合，使得TRAP成为能够发现响应于外部或遗传扰动的分子组分的有用方法。(C)2017爱思唯尔公司All rights reserved.
Organs and specific cell types execute specialized functions in multicellular organisms, in large part through customized gene expression signatures. Thus, profiling the transcriptomes of specific cell and tissue types remains an important tool for understanding how cells become specialized. Methodological approaches to detect gene expression differences have utilized samples from whole animals, dissected tissues, and more recently single cells. Despite these advances, there is still a challenge and a need in most laboratories to implement less invasive yet powerful cell-type specific transcriptome profiling methods. Here, we describe the use of the Translating Ribosome Affinity Purification (TRAP) method for C. elegans to detect cell type-specific gene expression patterns at the level of translating mRNAs. In TRAP, a ribosomal protein is fused to a tag (GFP) and is expressed under cell type-specific promoters to mark genetically defined cell types in vivo. Affinity purification of lysates of animals expressing the tag enriches for ribosome-associated mRNAs of the targeted tissue. The purified mRNAs are used for making cDNA libraries subjected to high-throughput sequencing to obtain genome-wide profiles of transcripts from the targeted cell type. The ease of exposing C. elegans to diverse stimuli, coupled with available cell type specific promoters, makes TRAP a useful approach to enable the discovery of molecular components in response to external or genetic perturbations. (C) 2017 Elsevier Inc. All rights reserved.