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

Area of Science:

• Immunology
• Genomics
• Cell Biology

Background:

• Eosinophils are crucial granulocytes with diverse roles in health and disease.
• Technical challenges have limited single-cell RNA sequencing (scRNA-seq) of eosinophils, hindering understanding of their heterogeneity and function.
• Gene regulatory networks governing eosinophil functions remain poorly understood.

Purpose of the Study:

• To develop and detail a novel, stress-free protocol for high-quality single-cell RNA capture from murine tissue-resident eosinophils.
• To identify distinct intestinal eosinophil subsets and elucidate their roles in colitis.
• To establish a method for functional genomic screening of eosinophils.

Main Methods:

• Developed a stress-free protocol combining magnetic enrichment with microwell-based scRNA capture (BD Rhapsody).
• Minimized shear stress and processing time for transcriptomic interrogation.
• Established genome-wide CRISPR pooled genetic screening for ex vivo-cultured bone marrow-derived eosinophils.

Main Results:

• Successfully captured high-quality single-cell transcriptomes from tissue-resident eosinophils, overcoming previous technical limitations.
• Identified distinct intestinal eosinophil subsets with specific roles in experimental colitis.
• Demonstrated the feasibility of functional genomic screening to probe eosinophil differentiation and maturation pathways.

Conclusions:

• The developed protocol enables comprehensive transcriptomic analysis of eosinophils, revealing cellular heterogeneity and function.
• This approach provides critical insights into eosinophil involvement in inflammatory diseases like colitis.
• The protocol is adaptable for studying other granulocytes, advancing research in immunology and disease pathogenesis.