Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing

Adipocyte size and fragility and commercial kit costs impose significant limitations on single -cell RNA sequencing of adipose tissue. Accordingly, we developed a workflow to isolate and sample-barcode nuclei from individual adipose tissue samples, integrating flow cytometry for quality control, counting, and precise nuclei pooling for direct loading onto the popular 103 Chromium controller. This approach can eliminate batch confounding, and significantly reduces poorquality nuclei, ambient RNA contamination, and droplet loading -associated reagent waste, resulting in pronounced improvements in information content and cost efficiency.