Optimized protocol for mouse footpad immune cell isolation for single-cell RNA sequencing and flow cytometry

Abstract

Summary: Single-cell RNA sequencing (scRNA-seq) requires the preparation of a highly viable single-cell suspension to get reliable sequencing results. Here, we present a protocol for isolating mouse footpad leukocytes while maintaining high viability. We describe steps for footpad collection, enzymatic tissue dissociation, leukocyte isolation and purification, and cell fixation and preservation. We then detail combinatorial barcoding, library preparation, scRNA-seq, and data analysis. Cells can be used to generate a complete molecular atlas at the single cell level. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Cell isolation
• Single Cell
• Flow Cytometry/Mass Cytometry
• RNAseq
• Immunology