1. Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing
- Author
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Anton Gulko, Adam Essene, Dylan Matthew Belmont-Rausch, Molly Veregge, Deepti Pant, Danielle Tenen, Benedicte Schultz Kapel, Margo P. Emont, Tune H. Pers, Evan D. Rosen, and Linus T. Tsai
- Subjects
Cell isolation ,Single Cell ,Cell separation/fractionation ,Flow Cytometry ,Genomics ,Sequencing ,Science (General) ,Q1-390 - Abstract
Summary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
- Published
- 2024
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