title: Sample 250
organism: Mus musculus
description: Protocols: Subcutaneous and visceral 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. 1,100 cells (550 RFP+ and 550 RFP-) at the concentration of 200 cells/µl were sorted by FACS directly into the C1 Single-Cell Auto Prep Array IFC 10-17µm chip (Fluidigm #100-5760) for 96 cells with the efficiency of ~10%. The presence of the cells was validated by microscopy visualisation (Olympus Cell Xcellence, objective: UPLFLN 10x/0.30). Single cell capture, lysis, reverse transcription and cDNA amplification were performed using the SMARTer cDNA synthesis kit (Clonetech) and Advantage2PCR kit (Clonetech) with addition of spike in (Ambion), within the IFC inside the the C1 Single-Cell Auto Prep system (Fluidigm) according to manufacturer's specifications. 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) utilising 96 dual barcoded indices. Library poolup was performed using AMPure XP beads (Agencourt Biosciences) and single cell libraries were sent for paired end sequencing at the Lausanne Genomics Technology Facility (GTF).