Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing

Summary: Adipocyte size and fragility and commercial kit costs impose significant limitations on single-cell RNA sequencing of adipose tissue. Accordingly, we developed a workflow to isolate and sample-barcode nuclei from individual adipose tissue samples, integrating flow cytometry for quality control, counting, and precise nuclei pooling for direct loading onto the popular 10× Chromium controller. This approach can eliminate batch confounding, and significantly reduces poor-quality nuclei, ambient RNA contamination, and droplet loading-associated reagent waste, resulting in pronounced improvements in information content and cost efficiency. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

A single-cell atlas of human and mouse white adipose tissue

none38noneMargo P. Emont, Christopher Jacobs, Adam L. Essene, Deepti Pant, Danielle Tenen, Georgia Colleluori, Angelica Di Vincenzo, Anja M. Jørgensen, Hesam Dashti, Adam Stefek, Elizabeth McGonagle, Sophie Strobel, Samantha Laber, Saaket Agrawal,Gregory P. Westcott, Amrita Kar, Molly L. Veregge, Anton Gulko, Harini Srinivasan, Zachary Kramer, Eleanna De Filippis, Erin Merkel, Jennifer Ducie, Christopher G. Boyd, William Gourash, Anita Courcoulas, Samuel J. Lin, Bernard T. Lee, Donald Morris, Adam Tobias, Amit V. Khera, Melina Claussnitzer, Tune H. Pers, Antonio Giordano, Orr Ashenberg, Aviv Regev, Linus T. Tsai & Evan D. RosenEmont, Margo P.; Jacobs, Christopher; Essene, Adam L.; Pant, Deepti; Tenen, Danielle; Colleluori, Georgia; DI VINCENZO, Angelica; Jørgensen, Anja M.; Dashti, Hesam; Stefek, Adam; Mcgonagle, Elizabeth; Strobel, Sophie; Laber, Samantha; Agrawal, Saaket; Westcott, Gregory P.; Kar, Amrita; Veregge, Molly L.; Gulko, Anton; Srinivasan, Harini; Kramer, Zachary; De Filippis, Eleanna; Merkel, Erin; Ducie, Jennifer; Boyd, Christopher G.; Gourash, William; Courcoulas, Anita; Lin, Samuel J.; Lee, Bernard T.; Morris, Donald; Tobias, Adam; Khera, Amit V.; Claussnitzer, Melina; Pers, Tune H.; Giordano, Antonio; Ashenberg, Orr; Regev, Aviv; Tsai &, Linus T.; Rosen, Evan D

Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology

Architecture of the outbred brown fat proteome defines regulators of metabolic physiology

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology