title: GSM9327876: Adipose tissue SVF eYFP+ cells, 12 weeks HFD; Mus musculus; RNA-Seq
library_construction_protocol: Adipose tissue was collected in PBS with 0.5% BSA (fatty-acid-free; Sigma A3803-100G) and kept on ice until ready to process. Tissue was minced using scalpels and digested in DMEM (Thermo Fisher, 41965039) containing 2% BSA, 12.5 mM HEPES, 0.2 mg/ml liberase TL (Sigma, 5401020001) and 1/100 DNase (Sigma, 10104159001) for 30min at 37 oC with constant agitation at 180 rpm. The digestion was stopped by adding one volume DMEM, and the sample centrifuged at 250xg for 10min to separate the stromovascular (SVF) and adipocyte fractions. Cell from the SVFs were strained through a 100 μm cell strainer, centrifuged at 300xg for 3min to pellet and red blood cells lysed by resuspending in ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4) for 2min. DMEM was added to stop the lysis, cells strained through a 40 μm cell strainer and centrifuged at 300xg for 3min to pellet. Cells were washed in FACS buffer (3% FBS, 2 mM EDTA) and pelleted at 300xg for 3min.  DAPI (10 ng/ml in FACS buffer) was added just before taking the samples for FACS. 100,000 events of live (DAPI-) eYFP+ cells were sortede per sample using a 100 μm nozzl into 500μl Post-sort buffer (0.04% BSA in PBS). Libraries were prepared using the 10x Single Cell 3' v3 kit according to manufacturer specifications.