title: GSM4875674: TN_1; Mus musculus; RNA-Seq
library_construction_protocol: Interscapular BAT was dissected, minced, and digested with a cocktail containing type 1 Collagenase (1.5 mg/mL; Worthington Biochemical), Dispase II (2.5 U/mL; STEMCELL Technologies), fatty acid free bovine serum albumin (2%; Gemini Bio Products) in Hanks' balanced salt's solution (Corning® Hank's Balanced Salt Solution, 1X with calcium and magnesium) for 45 minutes at 37°C with gentle shaking. Dissociated tissue was centrifuged at 500 g at 4°C for 10 minutes. The top adipocyte layer and the supernatant were gently removed, the SVF pellet was resuspended in 10 ml of 10% Fetal Bovine Serum (FBS) in DMEM, filtered through a 100 µm cell strainer into a fresh 50 ml tube, 38 and centrifuged at 500 g for 7 minutes. The red blood cell lysis was performed by resuspending the pellet in 2 ml sterile ACK (Ammonium-Chloride-Potassium) lysis buffer (ACK Lysing Buffer, Lonza) and incubating on ice for 5 minutes. The cells were then filtered through a 40 µm cell strainer, washed with 20 ml 10% FBS in DMEM, and centrifuged at 500 g for 7 minutes. The pellet was resuspended in 1 ml of 1.5% BSA in PBS. The dead cell removal was performed using Dead Cell Removal Kit (Miltenyi Biotec) according to the manufacturer's instructions. The cells were finally resuspended in 50- 100 µl of 1.5% BSA in PBS and kept on ice before immediately proceeding to single cell isolation. Single-cell RNA-seq libraries were obtained following the 10x Genomics recommended protocol, using the reagents included in the Chromium Single Cell 3′ v3 Reagent Kit.