Abstract Body: 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 changes depending on its anatomical location. Understanding cellular composition can give great insight into function. We had previously successfully performed single nucleus RNA-sequencing (snRNAseq) on brown fats, the thoracic aortic PVAT 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 yield of nuclei (< 200 nuclei/mg) and RNA for downstream single-nuclei analyses. Similar challenges were encountered when processing bovine WAT, which exhibits similar processing difficulties to those observed with human WAT. This study aimed to optimize and validate nuclei isolation protocols from human and mouse WAT in rat and bovine samples. This is important because it is WAT that surrounds vessels that govern total peripheral resistance. Protocols were evaluated based on a) the quantity of nuclei isolated, b) quality of nuclei determined by microscopic visualization, and c) total RNA content of the nuclei pellet. A protocol utilizing homogenization and wash buffers designed for nuclei isolation from frozen human WAT, as well as liquid nitrogen pulverization and Dounce homogenization of tissues, proved translatable to both rat and bovine WAT depots (rat retroperitoneal fat: 3100 nuclei/mg tissue; rat mesenteric perivascular adipose tissue: 2200 nuclei/mg tissue; bovine white fat; 1450 nuclei/mg tissue) with key modifications. We developed protocol modifications including lipid removal techniques and optimization of centrifugation speed, as well as the downstream applications and results of nuclei fixation and sequencing for all depots: rat WATs and BATs (both perivascular and non-perivascular) and bovine WAT. Collectively, we illustrate the importance of a robust nuclei isolation protocol that is generalizable across distinct adipose depot locations and species for the evaluation of single-nuclei transcriptomic profiles. This developed protocol should prove useful for snRNAseq of WAT from multiple species.