PME-seq – a whole-tissue RNA-seq toolkit for organism-wide studies of gene expression

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, University of Chicago researchers 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. The researchers report procedures to collect, store and lyse a dozen organ types using conditions compatible with the extraction of high-quality RNA. In addition, they 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.

Schematic overview of the PME-seq workflow

Fig. 1

 

a, Tissue collection to RNA extraction. Mice are transcardially perfused, and tissues of interest are collected and stored in an RNA-preserving solution. Tissues are subsequently lysed using Trizol and a bead-beater instrument to process up to 24 samples in parallel (except for liver and small intestine, which require different instrumentation due to their large size). Total RNA is extracted from tissue lysates using a high-throughput, magnetic bead-based protocol. b, Highly multiplexed RNA-seq library preparation. Total RNA is heat-fragmented and barcoded during RT with oligo(dT) priming. cDNA samples are then pooled and polymerized in their 3′ ends to add the Illumina Read 1 primer sequence. Finally, multiplexed libraries are enriched by PCR, which adds a second barcode, and the resulting dual-indexed libraries are sequenced.

Pandey S, Takahama M, Gruenbaum A, Zewde M, Cheronis K, Chevrier N. (2020) A whole-tissue RNA-seq toolkit for organism-wide studies of gene expression with PME-seq. Nat Protoc 15(4):1459-1483. [abstract]

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