GSM4985195: HFD 3; Mus musculus; RNA-Seq
For scRNA-seq, flow cytometry analysis, and flow cytometry sorting, mouse Epi/VAT were minced and digested in 4-6 mg/ml collagenase I, (Sigma-Aldrich), 4-6 mg/ml collagenase II (Roche), and 1% BSA (Sigma-Aldrich) in Hanks Balanced Salt Solution (Sigma-Aldrich) at 37°C for 45 min with agitation (180 rpm), then strained through a 70-μm strainer in PBS with 0.1% BSA. The cell suspension was centrifuged at 300xg for 5 min, the supernatant removed, and the cell pellet resuspended in PBS with 0.1% BSA. Red blood cell lysis was performed (BioLegend), followed by wash and centrifugation (300g, 5 min), resuspension in PBS, and straining through a 40μm filter. For scRNA-seq experiment, the SVF suspension was blocked in the anti-mouse CD16/32 antibody (BioLegend) for 30 min at 4°C followed by antibody incubation with CD11b-Alexa Fluor 488 and CD45-APC (BioLegend) for 30 at 4°C. Cells were then washed twice with PBS and sorted using fluorescence-activated cell sorting (FACS) (SY3200 Synergy, Sony Biotechnology). RNA libraries were prepared for sequencing using standard Illumina protocols