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

Protocol to perform polysome profiling in primary differentiating murine adipocytes

STAR Protoc. 2025 Apr 30;6(2):103799. doi: 10.1016/j.xpro.2025.103799. Online ahead of print.

Authors

Mirian Krystel De Siqueira¹, Zaynab Nouhi², Yutian Zhao¹, Siqi Wang³, Xinshu Xiao¹, Xia Yang⁴, Laura Hulea⁵, Claudio J Villanueva⁶

Affiliations

¹ Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Maisonneuve-Rosemont Hospital Research Centre, Montréal, QC H1T 2M4, Canada.
³ 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.
⁴ 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.
⁵ 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. Electronic address: laura.hulea@umontreal.ca.
⁶ 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. Electronic address: cvillanueva@ucla.edu.

Abstract

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.

Keywords: Cell Biology; Gene Expression; Metabolism; RNA-seq.