Dataset Information

Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling.

ABSTRACT: 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.

SUBMITTER: Whytock KL

PROVIDER: S-EPMC9876942 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling.

Whytock Katie L KL Divoux Adeline A Sun Yifei Y Hopf Meghan M Yeo Reichelle X RX Pino Maria F MF Yu GongXin G Smith Steven R SR Walsh Martin J MJ Sparks Lauren M LM

STAR protocols 20230120 1

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 gen ...[more]

PMID: 36853719

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Project description: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.

Dataset Information

Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling.

Publications

Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling.

Similar Datasets

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