GSM8206973
The fourth inguinal white adipose tissue (iWAT) depot from Ad-Lib or TAN mice were dissected and placed on a sterile 6-well tissue culture plate with ice-cold 1X DPBS. Excess liquid was removed from fat pads by blotting. Each tissue was cut and minced with scissors and then placed in 15 ml conical tubes containing digestion buffer (2 ml DPBS and Collagenase II at 3 mg/ml; Worthington Biochemical, Lakewood, NJ, USA) for 40 min of incubation at 37°C with gentle shaking at 100 rpm. Following tissue digestion, enzyme activity was stopped with 8 ml of resuspension media (DMEM/F12 with glutamax supplemented with 15%FBS and 1% pen/strep; Thermo Scientific, CA). The digestion mixture was passed through 100 μm cell strainer and centrifuged at 150 x g for 8 min at room temperature. To remove red blood cells, the pellet was resuspended and incubated in RBC lysis buffer (Thermo Scientific, CA) for 3 min at room temperature, followed by centrifugation at 150 x g for 8 min. The pellet was then resuspended in resuspension media and again spun down at 150 x g for 8 min. The cell pellet was resuspended in 1 ml of 0.01% BSA (in DPBS) and passed through a 40 μm cell strainer (Fisher Scientific, Hampton, NH, USA) to discard debris. Cell number was counted for 10X Genomics single cell application. To yield an expected recovery of 4000-7000 single cells, an estimated 10,000 single cells per channel were loaded onto Single Cell 3' Chip (10X Genomics, CA). The Single Cell 3' Chip was placed on a 10X Genomics instrument to generate single cell gel beads in emulsion (GEMs). Chromium Single Cell 3' v3 Library and Cell Bead Kits were used according to the manufacturer's instructions to prepare single cell RNA-Seq libraries.