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Summary: Automated single-cell dispensing is incompatible with white adipose tissue (WAT) due to lipid-laden adipocytes. Single-nuclei RNA-Seq permits transcriptional profiling of all cells from WAT. Human WAT faces unique technical challenges in isolating nuclei compared to rodent tissue due to greater extra-cellular matrix content and larger lipid droplets. In this protocol, we detail how to isolate nuclei from frozen subcutaneous human WAT for single-nuclei RNA-Seq.For complete information on the generation and use of this protocol, please refer to Whytock et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.