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Nuclei Isolation from Rat and Bovine White Adipose Tissue for Single-Nuclei RNA Sequencing 

ABSTRACT: Perivascular adipose tissue (PVAT) is a complex tissue that is increasingly recognized for its roles in vascular health and disease. The form and function of PVAT is different depending on species and anatomical location and understanding its cellular and molecular characteristics gives greater insights. We had previously successfully performed single nucleus RNA-sequencing (snRNAseq) on brown fat depots, the thoracic aortic PVAT (taPVAT) and subscapular brown adipose tissue (BAT), from Dahl SS rats.  However, application of the same nuclei isolation method to white adipose tissue (WAT) depots (perivascular and non-perivascular) from the same rat strain resulted in insufficient nuclei capture and low transcript numbers. These challenges were also encountered when processing WAT from cattle. While nuclei isolation methods have been developed and optimized for human and mouse WAT depots, they have not been evaluated across WAT depots from other species such as the cow and rat. Because these latter species are important models for cardiovascular and metabolic diseases, this study aimed to validate and optimize a nuclei isolation protocol for use with WAT from them. Protocols were evaluated based on (a) the quantity of nuclei isolated, (b) the quality of nuclei determined by microscopic visualization, and (c) the total number of detected transcripts and genes following snRNAseq. A modified protocol developed for human WAT, incorporating liquid nitrogen pulverization and Dounce homogenization of flash-frozen tissue was tested. This protocol, with key modifications for optimization, proved translatable to rat and cow WAT depots to improve nuclei yield (rat retroperitoneal fat: 3100 nuclei/mg tissue; rat mesenteric perivascular adipose tissue: 2200 nuclei/mg tissue; cow white fat 2050 nuclei/mg). Further analysis by snRNAseq, however, identified limitations. While cow WAT expressed nearly 1,720 median genes/cell, both rat white depots were significantly lower (mesPVAT: 189, RP fat: 294 median genes/cell), hindering downstream analyses in the rat tissue. These findings suggest that biological differences in adipose depots within and between species pose important challenges for the application of snRNAseq on rat WAT.

ORGANISM(S): Rattus norvegicus Bos taurus

PROVIDER: GSE320334 | GEO | 2026/04/01

REPOSITORIES: GEO

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Project description:Thoracic perivascular adipose tissue (PVAT) is a unique adipose depot that likely influences vascular function and susceptibility to pathogenesis in obesity and metabolic syndrome. Surprisingly, PVAT has been reported to share characteristics of both brown and white adipose, but a detailed direct comparison to interscapular brown adipose tissue (BAT) has not been performed. Here we show by full genome DNA microarray analysis that global gene expression profiles of PVAT are virtually identical to BAT, with equally high expression of Ucp-1, Cidea and other genes known to be uniquely or very highly expressed in BAT. PVAT and BAT also displayed nearly identical phenotypes upon immunohistochemical analysis, and electron microscopy confirmed that PVAT contained multilocular lipid droplets and abundant mitochondria. Compared to white adipose tissue (WAT), PVAT and BAT from C57BL/6 mice fed a high fat diet for 13 weeks had markedly lower expression of immune cell-enriched mRNAs, suggesting resistance to obesity-induced inflammation. Indeed, staining of BAT and PVAT for macrophage markers (F4/80, CD68) in obese mice showed virtually no macrophage infiltration, and FACS analysis of BAT confirmed the presence of very few CD11b+/CD11c+ macrophages in BAT (1.0%) in comparison to WAT (31%). In summary, murine PVAT from the thoracic aorta is virtually identical to interscapular BAT, is resistant to diet-induced macrophage infiltration, and thus may play an important role in protecting the vascular bed from thermal and inflammatory stress. 8-week-old male C57BL6/J mice were fed a normal (ND) or high fat diet (HFD) (Research Diets 12451, 45 kcal% fat) for 13 weeks. Mice were then euthanized and four different adipose depots were harvested for RNA analysis: perivascular fat from the lesser curvature of the aortic arch (PVAT), interscapular brown adipose (BAT), inguinal adipose tissue (SAT), and epididymal adipose tissue (VAT). 250 ng total RNA pooled from two mice was used for cDNA synthesis; 3 biological replicates per tissue and diet were performed for a total of 24 hybridizations.

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Nuclei Isolation from Rat and Bovine White Adipose Tissue for Single-Nuclei RNA Sequencing

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