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

Abstract

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.

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Fig. 1: Schematic overview of the PME-seq workflow.
Fig. 2: Anticipated results for RNA quality and library preparation.
Fig. 3: Patterns of mRNA expression and tissue-specific genes are similar between our protocol and existing mouse and human consortium data sets.

Data availability

The example datasets used in this protocol are available in the Gene Expression Omnibus under accession number GSE138103.

Code availability

All scripts are available at https://github.com/chevrierlab/PME-seq.

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Acknowledgements

We thank M. Kadoki, T. Matsui and S. Tay for helpful discussions. This work was supported by an Overseas Research Fellowship from the Astellas Foundation for Research on Metabolic Disorders (M.T.), an NIH Director’s New Innovator Award DP2 (AI145100), an Institutional Research Grant (IRG-16-222-56) from the American Cancer Society, the University of Chicago Medicine Comprehensive Cancer Center Support Grant (P30 CA14599), the Elliot and Ruth Sigal MRA Young Investigator Award (571146) and funds from the Pritzker School of Molecular Engineering (N.C.).

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Authors

Contributions

Conceptualization, N.C.; methodology and investigation, S.P., M.T., M.Z., K.C. and N.C.; formal analysis, A.G. and N.C.; writing, S.P., A.G. and N.C.; supervision and funding acquisition, N.C.

Corresponding author

Correspondence to Nicolas Chevrier.

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The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Zhou Xing and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Kadoki, M. et al. Cell 171, 398–413 (2017): https://doi.org/10.1016/j.cell.2017.08.024

Sage, P. T. et al. Nat. Immunol. 17, 1436–1446 (2016): https://doi.org/10.1038/ni.3578

Hou, S. et al. J. Exp. Med. 216, 605 (2019): https://doi.org/10.1084/jem.20181134

Integrated supplementary information

Supplementary Fig. 1 Setup of the peristaltic pump and associated materials to perform transcardial perfusion followed by tissue collections.

a, Materials for the peristaltic pump setup. Shown are all equipment, materials and associated catalog numbers. Before using a vacutainer, the opposite end of the butterfly needle is cut off and discarded. b, Peristaltic pump ready to use for perfusion. Tubing is linked to the vacutainer through a connector (Gilson, cat. no. F1179941). On the opposite side of the tubing, the tubing is connected to another piece of tubing by a connector (Gilson, cat. no. F117987), and the connection is wrapped with parafilm. c, Example setup for transcardial perfusion followed by tissue collection. In this case, Vesphene is used as a disinfectant for decontamination purposes when working with infectious materials.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Methods.

Reporting Summary

Supplementary Software 1

Suite of scripts (1 of 3) to use for RNA extraction automation using the Agilent Bravo liquid handling platform.

Supplementary Software 2

Suite of scripts (2 of 3) to use for RNA extraction automation using the Agilent Bravo liquid handling platform.

Supplementary Software 3

Suite of scripts (3 of 3) to use for RNA extraction automation using the Agilent Bravo liquid handling platform.

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Pandey, S., Takahama, M., Gruenbaum, A. et al. A whole-tissue RNA-seq toolkit for organism-wide studies of gene expression with PME-seq. Nat Protoc 15, 1459–1483 (2020). https://doi.org/10.1038/s41596-019-0291-y

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