GSM5669664: Sample 2_Aorta_HFD_Tie2Ctrl; Mus musculus; RNA-Seq
The aortas were harvested in PBS and their surrounding adipose tissues were removed. The aortas were then minced into pieces and digested in DPBS containing 1 mg/ml collagenases Type I (Gibco #17100-017), II (Gibco #17101-015) and IV (Gibco #17104-019) each for 30 minutes at 37oC water bath with mild mixing for a few times during the digestion. After the digestion, the cell suspension was filtered through a 70 μm cell strainer. The red blood cells were lysed with ACK Lysing buffer (Gibco # A10492-01) according to the instructions. Left lobes of the lungs were minced using scissors in a mixture of 0.5 mg/mL Collagenase H (Roche), 0.5 U/mL of Dispase II (Sigma), 1 U/mL DNase I (Thermo Scientific) and 1% (w/v) BSA (Biowest) in 4 mL of phosphate buffered saline (PBS) and incubated at 37ºC with constant agitation for 20 minutes. The slurry was then passed through a 70 µm nylon cell strainer (Corning) and the reaction was stopped with 10 mL of 10% heat-inactivated FBS in FACS-buffer (1% heat-inactivated FBS, 2 mM EDTA in PBS). Red blood cells were lysed by adding 1 mL of Red Blood Cell Lysis buffer (Sigma-Aldrich). The cells were incubated with Fc-blocker, as above, and the alive CD31+DAPI- cells were sorted using a BD Influx flow cytometer in single cell 1 drop setup with a 100 µm nozzle. After the FACS step,  lung ECs were resuspended in PBS containing 0.05% of 0.2 µm filter sterile BSA. Total bone marrow cells were isolated from both (left and right) femur of a mouse by flashing with the FACS buffer (PBS, supllemented with 0.05% BSA). We used the Chromium Single-Cell 3′RNA-sequencing system (10x Genomics, Pleasanton, CA) with the Reagent Kit v3.1 according to the manufacturer's instructions. Sample libraries were sequenced on the Illumina NovaSeq 6000 system using S2 flow cell (Illumina)