GSM8843030: VehicleAortaADV; Mus musculus; RNA-Seq
After isolation of the aorta from the thoracic aorta to the abdominal aorta, aorta adventitia layers were rapidly peeled out using micro forceps under the microscope on cold HBSS. In brief, the aortic adventitia was minced with microdissection scissors and then incubated with the enzyme mixture: LiberaseTM TL (Sigma-Aldrich, #540102001, 13 units/ml) and Elastase (Worthington, #LS006363, 48.4 units/ml) in HBSS (ThermoFisher, #14025092,), in the 37°C water bath for 20 min, followed by 37°C rotator for 40 min. After pipetting to prevent clumps, the cells were passed through a 40 µm strainer. Next, the cells were incubated with red blood cell lysis buffer (Abcam, # ab286858) for 5 min. The cells were rinsed with PBS with 0.04% BSA. 10 K cells were used for 3' mRNA library construction, and Single-cell RNA Sequencing was performed using the 10X Genomics Chromium platform at the UCLA Technology Center for Genomics and Bioinformatics. We performed paired-end sequencing to a read depth of 500 million, providing about 50,000 paired-end reads per cell.