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

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. Access through your institution Buy or

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ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS ACTIVE EOSINOPHILS REGULATE

HOST DEFENCE AND IMMUNE RESPONSES IN COLITIS Article Open access 12 December 2022 A FACS-BASED APPROACH TO OBTAIN VIABLE EOSINOPHILS FROM HUMAN ADIPOSE TISSUE Article Open access 06 August

2020 GUT MUCOSA DISSOCIATION PROTOCOLS INFLUENCE CELL TYPE PROPORTIONS AND SINGLE-CELL GENE EXPRESSION LEVELS Article Open access 14 June 2022 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

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CAS  PubMed  PubMed Central  Google Scholar  Download references 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. AUTHOR

INFORMATION Author notes * These authors contributed equally: Costanza Borrelli, Alessandra Gurtner. AUTHORS AND AFFILIATIONS * Department of Biosystems Science and Engineering, ETH Zürich,

Basel, Switzerland Costanza Borrelli & Andreas E. Moor * Institute of Experimental Immunology, University of Zürich, Zürich, Switzerland Alessandra Gurtner & Isabelle C. Arnold

Authors * Costanza Borrelli View author publications You can also search for this author inPubMed Google Scholar * Alessandra Gurtner View author publications You can also search for this

author inPubMed Google Scholar * Isabelle C. Arnold View author publications You can also search for this author inPubMed Google Scholar * Andreas E. Moor View author publications You can

also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHORS Correspondence to Isabelle C. Arnold or Andreas E. Moor. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER

REVIEW PEER REVIEW INFORMATION _Nature Protocols_ thanks Ariel Munitz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION

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SUPPLEMENTARY TABLE 1 Sequences for the primers used in the protocol. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this

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transcriptomic profiling and functional genomics of murine eosinophils. _Nat Protoc_ 19, 1679–1709 (2024). https://doi.org/10.1038/s41596-024-00967-3 Download citation * Received: 17 April

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