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Stress-free single-cell transcriptomic profiling and functional genomics of murine eosinophils

Nature Protocols (2024)Cite this article

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Abstract

Eosinophils are a class of granulocytes with pleiotropic functions in homeostasis and various human diseases. Nevertheless, they are absent from conventional single-cell RNA sequencing atlases owing to technical difficulties preventing their transcriptomic interrogation. Consequently, eosinophil heterogeneity and the gene regulatory networks underpinning their diverse functions remain poorly understood. We have developed a stress-free protocol for single-cell RNA capture from murine tissue-resident eosinophils, which revealed distinct intestinal subsets and their roles in colitis. Here we describe in detail how to enrich eosinophils from multiple tissues of residence and how to capture high-quality single-cell transcriptomes by preventing transcript degradation. By combining magnetic eosinophil enrichment with microwell-based single-cell RNA capture (BD Rhapsody), our approach minimizes shear stress and processing time. Moreover, we report how to perform genome-wide CRISPR pooled genetic screening in ex vivo-conditioned bone marrow-derived eosinophils to functionally probe pathways required for their differentiation and intestinal maturation. These protocols can be performed by any researcher with basic skills in molecular biology and flow cytometry, and can be adapted to investigate other granulocytes, such as neutrophils and mast cells, thereby offering potential insights into their roles in both homeostasis and disease pathogenesis. Single-cell transcriptomics of eosinophils can be performed in 2–3 d, while functional genomics assays may require up to 1 month.

Key points

  • This protocol describes a method for single-cell RNA sequencing of tissue-resident murine eosinophils and a procedure for genome-wide CRISPR pooled genetic screens in bone marrow-derived eosinophils.

  • The protocol is optimized to reduce RNA degradation during isolation by reducing shear stress and processing time using magnetic cell sorting techniques and microwell-based single-cell RNA capture.

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Fig. 1: A comparison of methods for isolation and RNA capture from murine eosinophils.
Fig. 2: A schematic overview of scRNASeq of murine eosinophils.
Fig. 3: A schematic overview of the CRISPRi screen in murine bone marrow-derived eosinophils.
Fig. 4: Purification of murine eosinophils from the blood: comparison of red blood cell lysis methods.
Fig. 5: Assessment of murine eosinophil enrichment and viability.
Fig. 6: Exemplary pipeline for scRNASeq data processing and eosinophil identification.
Fig. 7: Transduction, differentiation and sorting of murine BMSCs.

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Data availability

ScRNAseq data used to illustrate this protocol have been deposited at the Gene Expression Omnibus under the accession number GSE182001.

Code availability

The code used to analyze the data is available at https://github.com/Moors-Code/Eosinophils_scRNASeq.

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Acknowledgements

We thank K. Handler, D. Eletto, A. Ozga, F. Mhamedi Baccouche, M.-D. Hussherr and the Arnold laboratory for technical support and ideas. We are also thankful to the Single Cell Facility from BSSE and the Genomics Facility Basel for their help. This study was supported by an Eccellenza Professorial Fellowship from the Swiss National Science Foundation to I.C.A. (PCEFP3_187021) and A.E.M (PCEFP3_ 181249), by a Consolidator Grant from the Swiss National Science Foundation (TMCG-3_213857), by a TANDEM grant from the ISREC Foundation and by the Vontobel Foundation (1120/2022) to I.C.A., and by the Helmsley Charitable Trust grant no. 1903-03791 to A.E.M.

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Author notes

  1. These authors contributed equally: Costanza Borrelli, Alessandra Gurtner.

Authors and Affiliations

  1. Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

    Costanza Borrelli & Andreas E. Moor

  2. Institute of Experimental Immunology, University of Zürich, Zürich, Switzerland

    Alessandra Gurtner & Isabelle C. Arnold

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  1. Costanza Borrelli
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Contributions

C.B. and A.G designed the protocols, performed the experiments, analyzed the data and wrote the manuscript. I.C.A. and A.E.M supervised the study.

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Correspondence to Isabelle C. Arnold or Andreas E. Moor.

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

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Key reference using this protocol

Gurtner, A. et al. Nature 615, 151–157 (2023): https://doi.org/10.1038/s41586-022-05628-7

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary

Supplementary Table 1

Sequences for the primers used in the protocol.

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Borrelli, C., Gurtner, A., Arnold, I.C. et al. Stress-free single-cell transcriptomic profiling and functional genomics of murine eosinophils. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00967-3

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