Protocol to perform polysome profiling in primary differentiating murine adipocytes
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...
Saved in:
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-06-01
|
| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725002059 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850195568268148736 |
|---|---|
| author | Mirian Krystel De Siqueira Zaynab Nouhi Yutian Zhao Siqi Wang Xinshu Xiao Xia Yang Laura Hulea Claudio J. Villanueva |
| author_facet | Mirian Krystel De Siqueira Zaynab Nouhi Yutian Zhao Siqi Wang Xinshu Xiao Xia Yang Laura Hulea Claudio J. Villanueva |
| author_sort | Mirian Krystel De Siqueira |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-111b5de740bd4549a2a8db9028fed4cd |
| institution | OA Journals |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-111b5de740bd4549a2a8db9028fed4cd2025-08-20T02:13:43ZengElsevierSTAR Protocols2666-16672025-06-016210379910.1016/j.xpro.2025.103799Protocol to perform polysome profiling in primary differentiating murine adipocytesMirian Krystel De Siqueira0Zaynab Nouhi1Yutian Zhao2Siqi Wang3Xinshu Xiao4Xia Yang5Laura Hulea6Claudio J. Villanueva7Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USAMaisonneuve-Rosemont Hospital Research Centre, Montréal, QC H1T 2M4, CanadaDepartment of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USADepartment 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, ChinaDepartment of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USADepartment 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, USAMaisonneuve-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 authorDepartment 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 authorSummary: 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.http://www.sciencedirect.com/science/article/pii/S2666166725002059Cell BiologyRNA-seqMetabolismGene Expression |
| spellingShingle | Mirian Krystel De Siqueira Zaynab Nouhi Yutian Zhao Siqi Wang Xinshu Xiao Xia Yang Laura Hulea Claudio J. Villanueva Protocol to perform polysome profiling in primary differentiating murine adipocytes STAR Protocols Cell Biology RNA-seq Metabolism Gene Expression |
| title | Protocol to perform polysome profiling in primary differentiating murine adipocytes |
| title_full | Protocol to perform polysome profiling in primary differentiating murine adipocytes |
| title_fullStr | Protocol to perform polysome profiling in primary differentiating murine adipocytes |
| title_full_unstemmed | Protocol to perform polysome profiling in primary differentiating murine adipocytes |
| title_short | Protocol to perform polysome profiling in primary differentiating murine adipocytes |
| title_sort | protocol to perform polysome profiling in primary differentiating murine adipocytes |
| topic | Cell Biology RNA-seq Metabolism Gene Expression |
| url | http://www.sciencedirect.com/science/article/pii/S2666166725002059 |
| work_keys_str_mv | AT miriankrysteldesiqueira protocoltoperformpolysomeprofilinginprimarydifferentiatingmurineadipocytes AT zaynabnouhi protocoltoperformpolysomeprofilinginprimarydifferentiatingmurineadipocytes AT yutianzhao protocoltoperformpolysomeprofilinginprimarydifferentiatingmurineadipocytes AT siqiwang protocoltoperformpolysomeprofilinginprimarydifferentiatingmurineadipocytes AT xinshuxiao protocoltoperformpolysomeprofilinginprimarydifferentiatingmurineadipocytes AT xiayang protocoltoperformpolysomeprofilinginprimarydifferentiatingmurineadipocytes AT laurahulea protocoltoperformpolysomeprofilinginprimarydifferentiatingmurineadipocytes AT claudiojvillanueva protocoltoperformpolysomeprofilinginprimarydifferentiatingmurineadipocytes |