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

Eosinophils, a type of white blood cell, have long fascinated researchers due to their diverse roles in the body’s immune response. However, studying these enigmatic cells has proven challenging, as they are often overlooked in conventional single-cell RNA sequencing studies. Now, researchers have developed a groundbreaking protocol that sheds light on the complex world of eosinophils, revealing their heterogeneity and involvement in diseases like colitis. Developed by researchers at ETH Zürich, this stress-free method allows for the capture of high-quality single-cell transcriptomes from tissue-resident eosinophils, paving the way for a deeper understanding of their functions and regulatory networks.

Eosinophils play crucial roles in maintaining homeostasis and combating various human diseases, yet their heterogeneity and gene regulatory networks have remained elusive. Traditional methods have hindered researchers’ ability to study eosinophils at the single-cell level, limiting our understanding of their diverse functions. However, a newly developed protocol offers a solution to this challenge, enabling the enrichment and capture of eosinophil transcriptomes from murine tissues.

The protocol begins by enriching eosinophils from multiple tissue sites, such as the intestine, using a magnetic-based approach. This step ensures the isolation of pure eosinophil populations, free from contamination by other cell types. Next, the enriched eosinophils are subjected to single-cell RNA sequencing using microwell-based technology, such as BD Rhapsody, which minimizes shear stress and processing time, preserving the integrity of the transcripts.

A schematic overview of scRNA-Seq of murine eosinophils

Fig. 2

(1) Organs are collected and are kept at 4 °C until processing (maximum 1–2 h). (2) Tissues are minced into small pieces and incubated in a shaker at 37 °C for organ-specific enzymatic digestion. Enzyme mixes are described in the ‘Materials’ section. After 50 min, the suspension is filtered to eliminate debris. (3) To specifically label eosinophils, single-cell preparations are stained with Siglec-F PE antibody. (4) Anti-PE MicroBeads are added to the suspensions to magnetically label PE-conjugated cells. (5) After magnetic sorting, eosinophil viability and purity are assessed by Trypan blue staining and flow cytometry. (6) Organ-specific cell preparations are labeled with unique anti-CD45 DNA-barcoded hashing antibodies (sample tag). (7) Cells are loaded onto a BD Rhapsody cartridge and paired with cell capture beads. Cells are then lysed and mRNA transcripts are captured on magnetic oligonucleotide barcoded beads. Reverse transcription is performed on the beads. (8) After library preparation and Illumina sequencing, reads are demultiplexed on the basis of cell-specific barcodes and mapped to the reference genome. The resulting gene expression matrix can be used for downstream analysis.

Through this innovative approach, the researchers have uncovered distinct subsets of intestinal eosinophils and elucidated their roles in colitis, a type of inflammatory bowel disease. By analyzing the single-cell transcriptomes, researchers can identify key genes and pathways involved in eosinophil function and differentiation, providing valuable insights into their contribution to disease pathogenesis.

Furthermore, the protocol extends beyond transcriptomic analysis to include functional genomics assays, such as CRISPR pooled genetic screening. This powerful technique allows researchers to probe pathways required for eosinophil differentiation and maturation, offering a deeper understanding of their molecular mechanisms.

The development of this protocol represents a significant advancement in the study of eosinophils and other granulocytes. By overcoming technical challenges and providing a comprehensive framework for single-cell transcriptomics and functional genomics, researchers can now unravel the mysteries surrounding eosinophil heterogeneity and function.

Moreover, the protocol’s accessibility ensures that it can be adopted by any researcher with basic skills in molecular biology and flow cytometry. This democratization of technology opens doors for scientists worldwide to explore the roles of eosinophils and other granulocytes in health and disease.

The innovative protocol for single-cell RNA sequencing of eosinophils offers a transformative approach to studying these elusive immune cells. By providing a detailed methodology for enrichment and transcriptomic analysis, researchers can gain unprecedented insights into eosinophil heterogeneity and function. This pioneering work paves the way for future discoveries in immunology and opens new avenues for therapeutic interventions in diseases involving eosinophil dysregulation.

Borrelli C, Gurtner A, Arnold IC, Moor AE. (2024) Stress-free single-cell transcriptomic profiling and functional genomics of murine eosinophils. Nat Protoc [Epub ahead of print]. [abstract]

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