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Single-cell RNA and protein profiling of immune cells from the mouse brain and its border tissues

Nature Protocols volume 17, pages 2354–2388 (2022)Cite this article

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Abstract

Brain–immune cross-talk and neuroinflammation critically shape brain physiology in health and disease. A detailed understanding of the brain immune landscape is essential for developing new treatments for neurological disorders. Single-cell technologies offer an unbiased assessment of the heterogeneity, dynamics and functions of immune cells. Here we provide a protocol that outlines all the steps involved in performing single-cell multi-omic analysis of the brain immune compartment. This includes a step-by-step description on how to microdissect the border regions of the mouse brain, together with dissociation protocols tailored to each of these tissues. These combine a high yield with minimal dissociation-induced gene expression changes. Next, we outline the steps involved for high-dimensional flow cytometry and droplet-based single-cell RNA sequencing via the 10x Genomics platform, which can be combined with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and offers a higher throughput than plate-based methods. Importantly, we detail how to implement CITE-seq with large antibody panels to obtain unbiased protein-expression screening coupled to transcriptome analysis. Finally, we describe the main steps involved in the analysis and interpretation of the data. This optimized workflow allows for a detailed assessment of immune cell heterogeneity and activation in the whole brain or specific border regions, at RNA and protein level. The wet lab workflow can be completed by properly trained researchers (with basic proficiency in cell and molecular biology) and takes between 6 and 11 h, depending on the chosen procedures. The computational analysis requires a background in bioinformatics and programming in R.

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Fig. 1: Schematic overview of the protocol.
Fig. 2: Dissection of the subdural meninges, CP and dura mater.
Fig. 3: Sorting of brain immune cells for single-cell sequencing.
Fig. 4: scRNA-seq analysis of dissected border tissues or whole brain samples.
Fig. 5: CITE-seq analysis of GL261 tumor-infiltrating immune cells.
Fig. 6: CITE-seq analysis of CD45- dura mater cells.
Fig. 7: Level of ambient RNA contamination and percentage of doublets in different mouse brain tissues.
Fig. 8: HD flow cytometry analysis of whole brain and border samples.

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Data availability

The scRNA-seq data shown in Fig. 4a,b,e are deposited at GEO under GSE128855; Fig. 4f under GSE157480. The CITE-seq data shown in Fig. 5 are deposited at GEO under GSE163120. The gene–cell count matrices of all these datasets can also be downloaded at www.brainimmuneatlas.org. The CITE-seq data shown in Fig. 6 are deposited at GEO under GSE191075.

Code availability

The R codes that were used for scRNA-seq and CITE-seq analyses can be found at Github: https://github.com/Movahedilab/Mouse_brain_borders_NatureProtocols.

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Acknowledgements

This work was supported by Innoviris (Attract grant BB2B 2015−2), Fonds Wetenschappelijk Onderzoek (grant no. 1506316 N) to K.M., a VLAIO grant (no. ImmCyte HBC.2016.0889) to K.M. and JAVG. K.M. is a Collen-Franqui research professor. I.S. is supported by an FWO postdoctoral fellowship. H.V.H. is supported by an FWO predoctoral fellowship. We thank Y. Elkrim, G. van Isterdael, the VIB Flow Core and the VIB Nucleomics Core for technical assistance, VIB Tech Watch and the single-cell accelerator program for support regarding single-cell technologies.

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  1. These authors contributed equally: Isabelle Scheyltjens, Hannah Van Hove.

Authors and Affiliations

  1. Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium

    Isabelle Scheyltjens, Hannah Van Hove, Karen De Vlaminck, Daliya Kancheva, Jonathan Bastos, Mónica Vara-Pérez, Ana Rita Pombo Antunes, Jo A. Van Ginderachter & Kiavash Movahedi

  2. Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium

    Isabelle Scheyltjens, Hannah Van Hove, Karen De Vlaminck, Daliya Kancheva, Mónica Vara-Pérez, Ana Rita Pombo Antunes, Jo A. Van Ginderachter & Kiavash Movahedi

  3. Lab of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Brussels, Belgium

    Isabelle Scheyltjens, Hannah Van Hove, Karen De Vlaminck, Daliya Kancheva, Jonathan Bastos, Mónica Vara-Pérez & Kiavash Movahedi

  4. Laboratory of Myeloid Cell Biology in Tissue Damage and Inflammation, VIB Center for Inflammation Research, Ghent, Belgium

    Liesbet Martens & Charlotte L. Scott

  5. Laboratory of Myeloid Cell Biology in Tissue Homeostasis and Regeneration, VIB Center for Inflammation Research, Ghent, Belgium

    Liesbet Martens & Martin Guilliams

  6. Department of Biomedical Molecular Biology, Faculty of Sciences, Ghent University, Ghent, Belgium

    Liesbet Martens, Charlotte L. Scott & Martin Guilliams

  7. Data Mining and Modelling for Biomedicine, VIB Center for Inflammation Research, Ghent, Belgium

    Yvan Saeys & Niels Vandamme

  8. Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Ghent, Belgium

    Yvan Saeys & Niels Vandamme

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  1. Isabelle Scheyltjens
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Contributions

I.S., H.V.H., K.D.V. and K.M. developed and optimized the protocol with input and help from C.L.S., J.A.V.G., Y.S., M.G., N.V. I.S., H.V.H., K.D.V., J.B., M.V.P., A.R.P.A. and N.V. performed wet-lab work and acquired scRNA-seq and/or CITE-seq data. I.S., D.K., L.M. and Y.S. analyzed the data and/or generated analysis pipelines. I.S., H.V.H., D.K. and K.M. wrote the manuscript. K.M. conceptualized and directed the study.

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Correspondence to Kiavash Movahedi.

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Nature Protocols thanks Naomi Habib and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Van Hove, H. et al. Nat. Neurosci. 22, 1021–1035 (2019): https://doi.org/10.1038/s41593-019-0393-4

Shemer, A. et al. Immunity 54, 1033–1049.E7 (2020): https://doi.org/10.1016/j.immuni.2020.09.018

Pombo Antunes, A. R. et al. Nat. Neurosci. 24, 595–610 (2021): https://doi.org/10.1038/s41593-020-00789-y

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Scheyltjens, I., Van Hove, H., De Vlaminck, K. et al. Single-cell RNA and protein profiling of immune cells from the mouse brain and its border tissues. Nat Protoc 17, 2354–2388 (2022). https://doi.org/10.1038/s41596-022-00716-4

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