학술논문
A whole-tissue RNA-seq toolkit for organism-wide studies of gene expression with PME-seq
Document Type
Report
Author
Source
Nature Protocols. April 2020, Vol. 15 Issue 4, p1459, 25 p.
Subject
Language
English
ISSN
1754-2189
Abstract
Author(s): Surya Pandey [sup.1] , Michihiro Takahama [sup.1] , Adam Gruenbaum [sup.1] , Makda Zewde [sup.1] , Katerina Cheronis [sup.1] , Nicolas Chevrier [sup.1] Author Affiliations: (1) Pritzker School of [...]
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. This protocol describes collection, storage and lysis of different types of mouse organs for monitoring gene expression on a whole-organism scale. Procedures for high-throughput RNA extraction and multiplexed RNA sequencing library preparation are included.
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. This protocol describes collection, storage and lysis of different types of mouse organs for monitoring gene expression on a whole-organism scale. Procedures for high-throughput RNA extraction and multiplexed RNA sequencing library preparation are included.