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.

嗜酸性粒细胞是一类在体内平衡和各种人类疾病中具有多效功能的粒细胞。然而，由于技术上的困难阻碍了它们的转录组学研究，它们没有出现在传统的单细胞 RNA 测序图集中。因此，人们对嗜酸性粒细胞的异质性和支撑其不同功能的基因调控网络仍然知之甚少。我们开发了一种从鼠组织驻留嗜酸性粒细胞中捕获单细胞 RNA 的无压力方案，该方案揭示了不同的肠道亚群及其在结肠炎中的作用。在这里，我们详细描述了如何从多个驻留组织中富集嗜酸性粒细胞，以及如何通过防止转录物降解来捕获高质量的单细胞转录组。通过将磁性嗜酸性粒细胞富集与基于微孔的单细胞 RNA 捕获 (BD Rhapsody) 相结合，我们的方法最大限度地减少了剪切应力和处理时间。此外，我们报告了如何在离体条件化的骨髓来源的嗜酸性粒细胞中进行全基因组 CRISPR 混合遗传筛选，以功能性地探测其分化和肠道成熟所需的途径。这些协议可以由任何具有分子生物学和流式细胞术基本技能的研究人员执行，并且可以适用于研究其他粒细胞，例如中性粒细胞和肥大细胞，从而为它们在稳态和疾病发病机制中的作用提供潜在的见解。嗜酸性粒细胞的单细胞转录组学可以在 2-3 天内完成，而功能基因组学检测可能需要长达 1 个月的时间。