GSM8077869: Abdominal-Male; Mus musculus; RNA-Seq
30 minutes following SQ injection of 400 i.u of heparin 4 male and 4 female C57BL/6J mice were sacrificed and perfused with 10mL of Versene. Following perfusion, the carotid arteries and the aorta (from the aortic root to the iliac bifurcation) were harvested from the mouse and further dissected under a microscope to remove the adventitial layer. Subsequently the three vascular fragments: carotid arteries (right and left common not their branches), aortic arch (from the root to the subclavian branch), the thoracic aorta (below the subclavian up to the infra-renal branches, but excluding all branches) and the abdominal aorta (from the infra-renal branches to iliac birfucation, excluding all branches) were obtained, minced into small fragments and incubated under agitation into 500μL digestion buffer containing freshly prepared liberase solution (2.5% Liberase TH from frozen stock [Sigma Aldrich], 5 Kunitz Unit/mL DNase1, 1M HEPES IN 1X HBSS). All preparations were done at temperature except for the digestion, which was done at 37oC for approximately 20min. The suspension was neutralized with DMEM + 10%FBS and ran through a 40uM filter and then centrifuged for 5 min at 10,000 x g. Following centrifugation, the cell pellet was resuspended in in 0.4% BSA-PBS and assessed for viability prior to library preparation using trypan blue staining with the TC20 cell counter (BioRad). Only samples with more than 85% viability were used in the generation of libraries. Libraries were prepared using 10X Genomics Chromium™ Single Cell 3' Library & Gel Bead Kit v3, as per manufacturer's protocol. For the generation of single-cell gel beads in emulsion, cells were loaded on a Chromium single cell instrument (10x Genomics) with an estimated targeted cell recovery of ~5,000 to 10,000 cells, per library.