Visceral and Subcutaneous adipose tissue samples from lean and obese patients were evaluated by single nuclear RNA sequencing.  Nuclei were purified from frozen adipose tissue biopsies by FACS and libraries generated by 10X genomics platform.  

400-500mg samples of frozen human VAT or SAT were washed with sterile RNAse-free cold 1X DPBS and minced with a scalpel in a petri dish on ice. Samples were homogenized in a 15 mL precooled glass dounce homogenizer (10 strokes with pestel A followed by 15 strokes with pestel B) with 1.5mL of homogenization buffer [5mM MgCl2, 10mM Tris Buffer (pH 8.0), 25mM KCl, 250mM sucrose, 1X protease inhibitor, 1uM DTT, 0.4 U/uL Ribolock Rnase Inhibitor (40U/μL), 0.2U/uL Superasin (20U/uL), 0.1% triton X-100] and strained with pre-wet 100 μm and 40 μm filters into a 50mL conical tube. Next, each sample was transferred to two 1.5 mL pre-chilled microcentrifuge Rnase-free tubes and centrifuged at 500rcf, 4°C, 5 minutes. Supernatant was removed with a pipette and nuclei were stained with propidium iodide (PI) for sorting. Each sample was transferred into a pre-coated 5 mL polystyrene flow cytometry tube and sorted using a 100uM nozzle in a FACSAria III Cell Sorter. Sorting strategy included doublet discrimination and selection of intact nuclei by subgating on PI+ nuclei. PI+ nuclei was sorted directly into a 1.5 mL microcentrifuge tube containing 20uL of 1% BSA-DPBS and 0.2U/μl of Rnase Inhibitors. After nuclei isolation, samples were centrifuged at 100rcf, 4°C, 6 minutes, supernatant was pipetted off leaving approximately 50uL of resuspended nuclei. 3’ single nuclei libraries were generated using the 10X Genomics Chromium Controller.