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

• Published in: Nature protocols Vol. 19; no. 6; pp. 1679 - 1709
• Main Authors: Borrelli, Costanza, Gurtner, Alessandra, Arnold, Isabelle C., Moor, Andreas E.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.06.2024; Nature Publishing Group
• Subjects: 631/1647/2017; 631/1647/2163; 631/250/347; Analytical Chemistry; Animals; Biological Techniques; Biomedical and Life Sciences; Bone marrow; Colitis; Computational Biology/Bioinformatics; CRISPR; Degradation; Eosinophils; Eosinophils - metabolism; Flow cytometry; Gene Expression Profiling - methods; Gene sequencing; Genetic screening; Genomes; Genomics; Genomics - methods; Granulocytes; Heterogeneity; Homeostasis; Interrogation; Intestine; Leukocytes (eosinophilic); Leukocytes (granulocytic); Leukocytes (neutrophilic); Life Sciences; Mast cells; Mice; Mice, Inbred C57BL; Microarrays; Molecular biology; Organic Chemistry; Pathogenesis; Protocol; Ribonucleic acid; RNA; Shear stress; Single-Cell Analysis - methods; Transcriptome - genetics; Transcriptomes; Transcriptomics
• DOI: 10.1038/s41596-024-00967-3
• ISSN: 1754-2189; 1750-2799

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. This protocol presents a method for single-cell RNA sequencing of tissue-resident murine eosinophils, with a complementary method for CRISPR screening of bone marrow-derived eosinophils.