GSM5655544: aged adventitia; Mus musculus; RNA-Seq
In summary, mice were anaesthetized and perfused with 10mLs versene buffer through the left ventricle. The adventitia was removed and aorta dissected and cut open in versene buffer so that it laid like a flat sheet, exposing the endothelium. After versene washes, the aortae were bathed in 1X trypsin and incubated for 2X 5min at 37oC. Following this, the endothelium was gently removed using a microscalpel (EMS#72046-30) and repeat pipetting, now with sc-HBSS (containing 0.04%BSA and 2%FBS to inactivate the trypsin and Actinomycin D at 1ug/ml to block transcription) was applied this helped in removing the cells and obtaining a single cell suspension. The procedures were done under a dissecting microscope. Cells were pelleted and then treated with 1X RBC lysis buffer (eBioscience, #00-4333-57) for 1 minute and then washed twice with 0.04%BSA. Schematics of intimal cell isolation is shown in Figure 2A. To obtain enough cells, 6 (aged descending) or 8 (arch) C57BL/6 mice were used per library. scRNA-seq libraries were generated using the Chromium Single Cell 3' Library & Gel Bead Kit v2 (10X Genomics). Cells were loaded accordingly to 10X Genomics protocol with an estimated targeted cell recovery of 5000 cells.