title: GSM9052746: gWAT HFD-fed Young Male; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized using CO2 inhalation, and gonadal white adipose tissue (gWAT) was dissected. Tissue was finely minced and digested in 0.8 mg/ml Collagenase II (Worthington Biochemical) buffer containing 3% BSA, 1X penicillin/streptomycin, 1.2 mM CaCl₂, 1 mM MgCl₂, 0.8 mM ZnCl₂, and 15 mM HEPES for 40 minutes at 37°C with constant agitation. The digested suspension was centrifuged at 600g for 10 minutes at 4°C, and the SVF was filtered through 70 µm and 40 µm strainers. Red blood cell (RBC) lysis was performed as per manufacturer's instructions (eBioscience), followed by washing and resuspension in RPMI1640 supplemented with 10% FBS and 1X penicillin/streptomycin. Fresh cells were stained with Zombie Aqua live/dead dye (Biolegend) for 10 minutes at room temperature. Cells were sorted using a Bigfoot Spectral Cell Sorter (Thermo Fisher Scientific). Sorted cells were resuspended in RPMI medium with 10% FBS, equal number of cells from 3 mice per group were pooled and submitted to the University of Michigan Advanced Genomics Core for 3′ library preparation and sequencing. Briefly, cell counts were obtained using the Luna-FX7 Cell Counter (LogosBio). Libraries were prepared using the 10X Genomics Chromium Controller with 3′ v3.1 chemistry and Feature Barcoding technology for Cell Multiplexing, according to the manufacturer's instructions (10X Genomics). Library quality was assessed on the LabChip GXII HT (PerkinElmer), and concentrations were determined using Qubit (Thermo Fisher). Pooled libraries were sequenced using paired-end 28×10×10×151 bp reads on the Illumina NovaSeq XPlus. Demultiplexed FASTQ files were generated using Bcl2fastq2 (Illumina), and count matrices were generated using the CellRanger pipeline (10X Genomics).