Nuclei Isolation from Rat and Bovine White Adipose Tissue for snRNAseq on Parse Platform v1
Year: 2025
Type: preprint
Abstract: 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 protocols. These challenges were also encountered when processing WAT from cattle, which exhibits difficulties similar to those observed with human WAT. This study aimed to validate and optimize a nuclei isolation protocol for use with both rat and bovine WAT samples. This is important because it is WAT that surrounds the vessels that govern total peripheral resistance and is most associated with an increase in cardiovascular risk.Protocols were evaluated based on a) the quantity of nuclei isolated and b) quality of nuclei determined by microscopic visualization. A protocolutilizing liquid nitrogen pulverization and Dounce homogenization for flash-frozen tissue dissociation and homogenization and wash buffers designed for nuclei isolation of human WAT was tested. This protocol, with key modifications for optimization, proved translatable to 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). These protocol modifications included improved lipid removal techniques and optimization of centrifugation speed. 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.
Institution: Michigan State University
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Funder: National Institutes of Health
OpenAlex ID: W7133531071
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