Nuclei isolation from rat and cow white adipose tissues for single-nucleus RNA sequencing; rat WAT remains a challenge

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 are 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 Salt Sensitive rats. However, the 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 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 via 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, 3,100 nuclei/mg tissue; rat mesenteric perivascular adipose tissue, 2,200 nuclei/mg tissue; cow white fat, 2,050 nuclei/mg tissue). Further analysis via snRNAseq, however, identified limitations. While cow WAT expressed nearly 1,720 median genes/cell, both rat white depots were significantly lower [mesenteric PVAT (mesPVAT), 189; retroperitoneal (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.