A whole-tissue RNA-seq toolkit for organism-wide studies of gene expression with PME-seq.,Nature Protocols

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A whole-tissue RNA-seq toolkit for organism-wide studies of gene expression with PME-seq.
Nature Protocols ( IF 13.1 ) Pub Date : 2020-02-19 , DOI: 10.1038/s41596-019-0291-y
Surya Pandey ₁ , Michihiro Takahama ₁ , Adam Gruenbaum ₁ , Makda Zewde ₁ , Katerina Cheronis ₁ , Nicolas Chevrier ₁

Affiliation

The immune system operates at the scale of the whole organism in mammals. We currently lack experimental approaches to systematically track and study organism-wide molecular processes in mice. Here we describe an integrated toolkit for measuring gene expression in whole tissues, 3-prime mRNA extension sequencing, that is applicable to most mouse organs and any mouse model of interest. Further, the methods of RNA-seq described in this protocol are broadly applicable to other sample types beyond whole organs, such as tissue samples or isolated cell populations. We report procedures to collect, store and lyse a dozen organ types using conditions compatible with the extraction of high-quality RNA. In addition, we detail protocols to perform high-throughput and low-cost RNA extraction and sequencing, as well as downstream data analysis. The protocol takes 5 d to process 384 mouse organs from collecting tissues to obtaining raw sequencing data, with additional time required for data analysis and mining. The protocol is accessible to individuals with basic skills in (i) mouse perfusion and dissection for sample collection and (ii) computation using Unix and R for data analysis. Overall, the methods presented here fill a gap in our toolbox for studying organism-wide processes in immunology and physiology.

中文翻译：

一种全组织RNA-seq工具包，用于使用PME-seq对整个生物进行基因表达研究。

免疫系统以哺乳动物整个生物体的规模运作。我们目前缺乏实验方法来系统地追踪和研究小鼠体内的全生物分子过程。在这里，我们描述了一种用于测量整个组织中基因表达，3-prime mRNA延伸测序的集成工具包，该工具包适用于大多数小鼠器官和任何感兴趣的小鼠模型。此外，此方案中描述的RNA-seq方法可广泛应用于除整个器官以外的其他样品类型，例如组织样品或分离的细胞群。我们报告了使用与提取高质量RNA兼容的条件收集，储存和裂解多种器官类型的程序。此外，我们详细介绍了执行高通量和低成本RNA提取和测序以及下游数据分析的协议。该协议需要5天的时间来处理384个小鼠器官，从收集组织到获得原始测序数据，以及数据分析和挖掘所需的额外时间。具有以下基本技能的个人可以访问该协议：（i）进行鼠标灌注和解剖以收集样本，以及（ii）使用Unix和R进行数据分析的计算。总体而言，此处介绍的方法填补了我们研究免疫和生理学全有机体过程的工具箱中的空白。

更新日期：2020-02-19

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