RNA-seq of macrophage subsets and adipose tissue progenitors in the epididymal adipose tissue of chow- and high-fat diet-fed WT and Ccr2-deficient animals (C3H background)
RNA-seq of macrophage subsets and adipose tissue progenitors in the epididymal adipose tissue of chow- and high-fat diet-fed WT and Ccr2-deficient animals (C3H background)
Mouse epidydimal adipose tissue was harvested and digested in Hanks Balanced Salt Solution (HBSS,no calcium and magnesium) containing fetal bovine serum (FBS,3%) and collagenase D (2.5 mg/ml,Sigma-Aldrich) for 30 minutes at 37°C under agitation. After homogenization, the suspension is washed in PBS buffer containing bovine serum albumin (1%) and EDTA (0.5%). After centrifugation, the adipocyte fraction is discarded and the stroma vascular fraction passed through a 100 µm filter before staining. Cell suspensions were stained with appropriate antibodies for 30 min on ice. Cell sorting was performed on a BD FACSAria II™ cell sorter. RNAs from sorted cells were prepared using the RNeasy Plus micro kit (Qiagen). cDNA libraries were generated using the KAPA mRNA HyperPrep kit (Roche).