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

Costanza Borrelli, Alessandra Gurtner, Isabelle C. Arnold, Andreas E. Moor

Published: 2024-03-19 DOI: 10.1038/s41596-024-00967-3

CRISPR screening

single-cell RNA sequencing

eosinophils

granulocytes

intestinal subsets

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Sections

Figures

References

Introduction
Materials
- Stress-free scRNASeq of tissue resident eosinophils
- Pooled CRISPRi screen in bone-marrow derived eosinophils
Procedure 1: single-cell profiling of tissue resident eosinophils
Procedure 2: pooled CRISPRi screen in bone-marrow derived eosinophils
Troubleshooting
Timing
- Single-cell profiling of tissue resident eosinophils
- Pooled CRISPRi screen in bone marrow-derived eosinophils
Anticipated results
- Reporting summary
Acknowledgements

Denisenko, E. et al. Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows. Genome Biol. 21, 130 (2020).
Slyper, M. et al. A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nat. Med. 26, 792–802 (2020).
Heng, T. S. P. et al. The Immunological Genome Project: networks of gene expression in immune cells. Nat. Immunol. 9, 1091–1094 (2008).
Schulte-Schrepping, J. et al. Severe COVID-19 is marked by a dysregulated myeloid cell compartment. Cell 182, 1419–1440.e23 (2020).
Grieshaber-Bouyer, R. et al. The neutrotime transcriptional signature defines a single continuum of neutrophils across biological compartments. Nat. Commun. 12, 2856 (2021).
Ballesteros, I. et al. Co-option of neutrophil fates by tissue environments. Cell 183, 1282–1297.e18 (2020).
Huang, J. et al. Single-cell transcriptome profiling reveals neutrophil heterogeneity and functional multiplicity in the early stage of severe burn patients. Front. Immunol. 12, 792122 (2021).
Miyake, K. et al. Single cell transcriptomics clarifies the basophil differentiation trajectory and identifies pre-basophils upstream of mature basophils. Nat. Commun. 14, 2694 (2023).
Wechsler, M. E. et al. Eosinophils in health and disease: a state-of-the-art review. Mayo Clin. Proc. 96, 2694–2707 (2021).
Gurtner, A. et al. Active eosinophils regulate host defence and immune responses in colitis. Nature 615, 151–157 (2023).
Shah, K., Ignacio, A., McCoy, K. D. & Harris, N. L. The emerging roles of eosinophils in mucosal homeostasis. Mucosal Immunol. 13, 574–583 (2020).
Mao, H. et al. Mechanisms of Siglec-F-induced eosinophil apoptosis: a role for caspases but not for SHP-1, Src kinases, NADPH oxidase or reactive oxygen. PLoS ONE 8, e68143 (2013).
Knuplez, E. et al. Frontline science: superior mouse eosinophil depletion in vivo targeting transgenic Siglec-8 instead of endogenous Siglec-F: mechanisms and pitfalls. J. Leukoc. Biol. 108, 43–58 (2020).
Shamri, R. et al. CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules. FASEB J. 26, 2084–2093 (2012).
Rosenberg, H. F. Eosinophil-derived neurotoxin (EDN/RNase 2) and the mouse eosinophil-associated RNases (mEars): expanding roles in promoting host defense. Int. J. Mol. Sci. 16, 15442–15455 (2015).
Shum, E. Y., Walczak, E. M., Chang, C. & Christina Fan, H. in Single Molecule and Single Cell Sequencing. (ed. Suzuki, Y.) 63–79 (Springer, 2019).
Fan, H. C., Fu, G. K. & Fodor, S. P. A. Combinatorial labeling of single cells for gene expression cytometry. Science 347, 1258367 (2015).
Dent, L. A., Strath, M., Mellor, A. L. & Sanderson, C. J. Eosinophilia in transgenic mice expressing interleukin 5. J. Exp. Med. 172, 1425–1431 (1990).
Dyer, K. D. et al. Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow. J. Immunol. 181 4004–4009 (2008).
Wang, L. et al. Single-cell transcriptomic analysis reveals the immune landscape of lung in steroid-resistant asthma exacerbation. Proc. Natl Acad. Sci. USA 118, e2005590118 (2021).
Tang, W. et al. Single-cell RNA-sequencing in asthma research. Front. Immunol. 13, 988573 (2022).
Smillie, C. S. et al. Intra- and inter-cellular rewiring of the human colon during ulcerative colitis. Cell 178, 714–730.e22 (2019).
Colic, M. & Hart, T. Common computational tools for analyzing CRISPR screens. Emerg. Top. Life Sci. 5, 779–788 (2021).
Martin, J. C. et al. Single-cell analysis of Crohn’s disease lesions identifies a pathogenic cellular module associated with resistance to anti-TNF therapy. Cell 178, 1493–1508.e20 (2019).
Elmentaite, R. et al. Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohn’s disease. Dev. Cell 55, 771–783.e5 (2020).
Kong, L. et al. The landscape of immune dysregulation in Crohn’s disease revealed through single-cell transcriptomic profiling in the ileum and colon. Immunity 56, 444–458.e5 (2023).
Fawkner-Corbett, D. et al. Spatiotemporal analysis of human intestinal development at single-cell resolution. Cell 184, 810–826.e23 (2021).
Ho, Y.-T. et al. Longitudinal single-cell transcriptomics reveals a role for Serpina3n-mediated resolution of inflammation in a mouse colitis model. Cell Mol. Gastroenterol. Hepatol. 12, 547–566 (2021).
Cui, A. et al. Single-cell atlas of the liver myeloid compartment before and after cure of chronic viral hepatitis. J. Hepatol. http://doi.org/10.1016/j.jhep.2023.02.040 (2023).
Salcher, S. et al. High-resolution single-cell atlas reveals diversity and plasticity of tissue-resident neutrophils in non-small cell lung cancer. Cancer Cell 40, 1503–1520.e8 (2022).
Schwarzfischer, M. et al. TiO2 nanoparticles abrogate the protective effect of the Crohn’s disease-associated variation within the PTPN22 gene locus. Gut http://doi.org/10.1136/gutjnl-2021-325911 (2022).
Gao, C., Zhang, M. & Chen, L. The comparison of two single-cell sequencing platforms: BD Rhapsody and 10x Genomics Chromium. Curr. Genomics 21, 602–609 (2020).
Mayer, A. T. et al. A tissue atlas of ulcerative colitis revealing evidence of sex-dependent differences in disease-driving inflammatory cell types and resistance to TNF inhibitor therapy. Sci. Adv. 9, eadd1166 (2023).
Lafzi, A. et al. Identifying spatial co-occurrence in healthy and inflamed tissues (ISCHIA). Mol. Syst. Biol. 20, 98–119 (2024).
Bock, C. et al. High-content CRISPR screening. Nat. Rev. Methods Prim. 2, 1–23 (2022).
Shi, H., Doench, J. G. & Chi, H. CRISPR screens for functional interrogation of immunity. Nat. Rev. Immunol. http://doi.org/10.1038/s41577-022-00802-4 (2022).
Parnas, O. et al. A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell 162, 675–686 (2015).
Yeung, A. T. Y. et al. A genome-wide knockout screen in human macrophages identified host factors modulating Salmonella infection. mBio 10, e02169–19 (2019).
Lai, Y. et al. High-throughput CRISPR screens to dissect macrophage-shigella interactions. mBio 12, e0215821 (2021).
Cortez, J. T. et al. CRISPR screen in regulatory T cells reveals modulators of Foxp3. Nature 582, 416–420 (2020).
Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Morgens, D. W. et al. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nat. Commun. 8, 15178 (2017).
Rossi, A. et al. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature 524, 230–233 (2015).
Gurtner, A., Gonzalez-Perez, I. & Arnold, I. C. Intestinal eosinophils, homeostasis and response to bacterial intrusion. Semin. Immunopathol. 43, 295–306 (2021).
Mair, F. et al. A targeted multi-omic analysis approach measures protein expression and low-abundance transcripts on the single-cell level. Cell Rep. 31, 107499 (2020).
Lee, J. J. et al. Human versus mouse eosinophils: ‘that which we call an eosinophil, by any other name would stain as red’. J. Allergy Clin. Immunol. 130, 572–584 (2012).
Chu, D. K. et al. Indigenous enteric eosinophils control DCs to initiate a primary Th2 immune response in vivo. J. Exp. Med. 211, 1657–1672 (2014).
Feng, Y.-H. & Mao, H. Expression and preliminary functional analysis of Siglec-F on mouse macrophages. J. Zhejiang Univ. Sci. B 13, 386–394 (2012).
DelGiorno, K. E. et al. Tuft cell formation reflects epithelial plasticity in pancreatic injury: implications for modeling human pancreatitis. Front. Physiol. 11, 88 (2020).
Percopo, C. M., Limkar, A. R., Sek, A. C. & Rosenberg, H. F. in Eosinophils: Methods and Protocols. (ed. Walsh, G. M.) 49–58 (Springer, 2021).
Olbrich, C. L., Larsen, L. D. & Spencer, L. A. Assessing phenotypic heterogeneity in intestinal tissue eosinophils. Methods Mol. Biol. 2241, 243–255 (2021).
Munoz, N. M. & Leff, A. R. Highly purified selective isolation of eosinophils from human peripheral blood by negative immunomagnetic selection. Nat. Protoc. 1, 2613–2620 (2006).
Ide, M., Weiler, D., Kita, H. & Gleich, G. J. Ammonium chloride exposure inhibits cytokine-mediated eosinophil survival. J. Immunol. Methods 168, 187–196 (1994).
Stoeckius, M. et al. Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol. 19, 224 (2018).
McGinnis, C. S. et al. MULTI-seq: sample multiplexing for single-cell RNA sequencing using lipid-tagged indices. Nat. Methods 16, 619–626 (2019).
Handler, K. et al. Fragment-sequencing unveils local tissue microenvironments at single-cell resolution. Nat. Commun. 14, 7775 (2023).
Parekh, S., Ziegenhain, C., Vieth, B., Enard, W. & Hellmann, I. zUMIs—a fast and flexible pipeline to process RNA sequencing data with UMIs. Gigascience 7, giy059 (2018).
Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife 5, e19760 (2016).
Joung, J. et al. Genome-scale CRISPR–Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828–863 (2017).
Lu, T. X. & Rothenberg, M. E. Bone marrow derived eosinophil cultures. Bio. Protoc. 4, e1161–e1161 (2014).
Hudak, S. et al. FLT3/FLK2 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units. Blood 85, 2747–2755 (1995).
Metcalf, D., Mifsud, S. & Di Rago, L. Stem cell factor can stimulate the formation of eosinophils by two types of murine eosinophil progenitor cells. Stem Cells 20, 460–469 (2002).
Gouvarchin Ghaleh, H. E., Bolandian, M., Dorostkar, R., Jafari, A. & Pour, M. F. Concise review on optimized methods in production and transduction of lentiviral vectors in order to facilitate immunotherapy and gene therapy. Biomed. Pharmacother. 128, 110276 (2020).
Wang, B. et al. Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute. Nat. Protoc. 14, 756–780 (2019).
Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).
Xie, X. et al. Single-cell transcriptome profiling reveals neutrophil heterogeneity in homeostasis and infection. Nat. Immunol. 21, 1119–1133 (2020).

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

Supplementary information

Supplementary Information

Reporting Summary

Supplementary Table 1

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