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Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses.
Yang, Chih-Hsiang; Longinotto, John; Panzeri, Ilaria; Arrigoni, Laura; Wegert, Vanessa; Heyne, Steffen; Seifert, Gabriel; Lempradl, Adelheid; Bönisch, Ulrike; Pospisilik, John Andrew.
Afiliación
  • Yang CH; Van Andel Institute; Max Planck Institute of Immunobiology and Epigenetics.
  • Longinotto J; Max Planck Institute of Immunobiology and Epigenetics.
  • Panzeri I; Van Andel Institute; Max Planck Institute of Immunobiology and Epigenetics.
  • Arrigoni L; Max Planck Institute of Immunobiology and Epigenetics.
  • Wegert V; Van Andel Institute.
  • Heyne S; Max Planck Institute of Immunobiology and Epigenetics.
  • Seifert G; Department of General and Visceral Surgery, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg.
  • Lempradl A; Van Andel Institute; Max Planck Institute of Immunobiology and Epigenetics.
  • Bönisch U; Max Planck Institute of Immunobiology and Epigenetics.
  • Pospisilik JA; Van Andel Institute; Max Planck Institute of Immunobiology and Epigenetics; andrew.pospisilik@vai.org.
J Vis Exp ; (184)2022 06 16.
Article en En | MEDLINE | ID: mdl-35786676
Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Adipocitos Blancos / Transcriptoma Límite: Animals Idioma: En Revista: J Vis Exp Año: 2022 Tipo del documento: Article

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Adipocitos Blancos / Transcriptoma Límite: Animals Idioma: En Revista: J Vis Exp Año: 2022 Tipo del documento: Article