title: GSM9013388: Aorta-derived cells, Rep 2, CMO multiplex. Groups: Ctrl2_4wks, ImP2_4wks, Ctrl2_8wks, ImP2_8wks.; Mus musculus; OTHER
library_construction_protocol: After treatment, mice were perfused with 10 mL of  PBS via cardiac puncture to remove blood contamination from vascular tissue. The aortas were kept in cold Dulbecco's Modified Eagle Medium (DMEM) to be digested. Perivascular fat was removed and the thoracic aorta with arch was opened longitudinally and cut into smaller pieces that were incubated for 30 min at 37ºC in a water bath in digestion buffer (Collagenase A (25 mg/ml), (Roche/Sigma #10103586001). Dispase II (25 mg/ml), (Roche/Sigma #04942078001), DNase I (250 μg/ml) (Roche/Sigma #10104159001) elastase (25 μg/ml) and Libersase TL (0.2 Wünsch units/ml, # 5401119001). After incubation time the digested tissue was mixed by pipetting, filtered through a 70 μm strainer and spinned at 400 x g 5 min at 4ºC. To obtain enough cell numbers, 3 aortae per condition were pooled. Each sample was labeled with a cell multiplexing oligo (CMO) using the CMO labeling for Single Cell RNA Sequencing Protocol from 10x Genomics. Cells labeled with CMOs were washed, labelled with SytoxGreen and Hoechst to ensure cell viability and live cells sorted with a FACs Aria cell sorter. Samples were pooled and single-cell RNA-seq was performed following the Chromium Next GEM Single Cell 3' v3.1 with feature barcode technology for cell multiplexing protocol (10x Genomics). Briefly, cells were first counted and checked their viability using the Countess 3 cell counter (Thermofisher). Next, cells were loaded onto a 10x Genomics chip of the Chromium Controller (10x Genomics).