title: Sample 4
organism: Mus musculus
description: Protocols: Subcutaneous adipose tissue depots were dissected from male and female 8-12-week-old Pref1-RFP mice into ice cold PBS. The tissue was finely minced using scissors, transferred into collagenase (Sigma-Aldrich #C6885-1G, 2 mg/ml of collagenase buffer (25 mM NaHCO3, 12 mM KH2PO4, 1.2 m MgSO4, 4.8 mM KCl, 120 mM NaCl, 1.4 mM CaCl2, 5 mM Glucose, 2.5% BSA, pH = 7.4)) and incubated for 1 hour at 37_C under agitation. Following a 1 min centrifugation at 400g, at room temperature (RT), floating mature lipid-filled adipocytes were aspirated and the suspension was filtered through a 40 µm cell strainer to ensure a single cell preparation. Next, a red blood cell (RBC) lysis was performed by incubating the pelleted cells in the RBC lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 5 min, followed by two washes (5 min centrifugations at 400g, RT) with FACS buffer (PBS with 3% foetal bovine serum (FBS) (Gibco #10270-106), 1mM EDTA, 1 % penicillin/streptavidin (P/S) (Gibco #15140122)). The isolated single cell suspension was diluted to 0.75 or 1 * 107 cells/ml with FACS buffer (PBS with 3% FBS, 1mM EDTA, 1% P/S and the following fluorophore-conjugated antibodies were added (in titration-determined quantities): anti-mouse CD31-AF488, anti-mouse CD45-AF488, anti-mouse TER119-AF488 (all BioLegend #303110, #304017 and #116215 respectively) for selecting the Lin- population, anti-mouse SCA1-PE-Cy7 (BioLegend #122513), anti-mouse CD34-BV421 (BioLegend #119321) and anti-mouse CD29- PerCP-efluor710 (eBiosciences #46-0291) to enrich the Lin- population with ASPCs. Single cell capture, lysis, reverse transcription and cDNA amplification were performed using the SMARTer cDNA synthesis kit (Clonetech) and Advantage2PCR kit (Clonetech). Harvested cDNA from single cells after C1-Autoprep system run were used to make single cell libraries using Nextera XT DNA sample preparation kit (Illumina).