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Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications.

Journal of visualized experiments : JoVE (2020-07-01)
Gabrielle J Benitez, Kosaku Shinoda
ZUSAMMENFASSUNG

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

MATERIALIEN
Produktnummer
Marke
Produktbeschreibung

Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
Magnesiumchlorid -Lösung, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Albumin -Lösung, 10% in DPBS, low endotoxin, fatty acid free, suitable for cell culture, sterile-filtered
Sigma-Aldrich
Calciumchlorid -Lösung, BioUltra, for molecular biology, ~1 M in H2O
Roche
Dispase® II (neutrale Protease, Klasse II), lyophilized, from bacterial, Roche, pkg of 5 × 1 g