Protocol to perform polysome profiling in primary differentiating murine adipocytes

Mirian Krystel De Siqueira; Zaynab Nouhi; Yutian Zhao; Siqi Wang; Xinshu Xiao; Xia Yang; Laura Hulea; Claudio J. Villanueva

doi:10.1016/j.xpro.2025.103799

STAR Protocols (Jun 2025)

Protocol to perform polysome profiling in primary differentiating murine adipocytes

Mirian Krystel De Siqueira,
Zaynab Nouhi,
Yutian Zhao,
Siqi Wang,
Xinshu Xiao,
Xia Yang,
Laura Hulea,
Claudio J. Villanueva

Affiliations

Mirian Krystel De Siqueira: Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA
Zaynab Nouhi: Maisonneuve-Rosemont Hospital Research Centre, Montréal, QC H1T 2M4, Canada
Yutian Zhao: Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA
Siqi Wang: Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA; CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai 200031, China
Xinshu Xiao: Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA
Xia Yang: Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90025, USA
Laura Hulea: Maisonneuve-Rosemont Hospital Research Centre, Montréal, QC H1T 2M4, Canada; Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3C 3J7, Canada; Département de Médecine, Université de Montréal, Montréal, QC H3C 3J7, Canada; Corresponding author
Claudio J. Villanueva: Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Corresponding author

DOI: https://doi.org/10.1016/j.xpro.2025.103799
Journal volume & issue: Vol. 6, no. 2
p. 103799

Abstract

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Summary: Here, we present a protocol for polysome profiling in differentiating adipocytes from the mouse stromal vascular fraction. We describe steps for lysate preparation, ultracentrifugation through sucrose gradients, mRNA isolation, RNA sequencing, and motif enrichment. This protocol enables the analysis of actively translating mRNAs and translational gene regulation by isolating polysome-bound mRNA, revealing insights into protein synthesis during adipogenesis or stimuli responses. Applications include studying translational control in adipocytes, metabolic diseases, and obesity, linking translational regulation to cellular and metabolic phenotypes.For complete details on the use and execution of this protocol, please refer to De Siqueira et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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