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

Abstract

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 10× Chromium controller. This approach can eliminate batch confounding, and significantly reduces poor-quality nuclei, ambient RNA contamination, and droplet loading-associated reagent waste, resulting in pronounced improvements in information content and cost efficiency.

Keywords: Cell isolation; Cell separation/fractionation; Flow Cytometry; Genomics; Molecular Biology; RNA-seq; Sequencing; Single Cell.