GSM8077092: Control_16wk_3_nontdT; Mus musculus; RNA-Seq
Aortic tissue was dissected from the aortic root to the brachiocephalic artery. Protocol follows as such:1) Prepare the Enzyme Solution (on ice): Final (working) concentration of Liberase is 2 units/mL Final (working) concentration of Elastase is 2 units/mL For 1mL of enzyme solution: Liberase TM (26 units/mL)77  uL Elastase (125 units/mL)16  uL HBSS907 uL Total1   mL 2) Place enzyme mixture on ice while aorta is being harvested 3) Perfuse mouse with ~10mL PBS via LV puncture 4) Harvest aortic root and open longitudinally. Wash with PBS x3 in 12-well dish. Keep on ice while collecting from multiple mice. 5) Move root to 12-well dish with 1mL of enzyme mixture. 6) Incubate in 37C incubator for ~30-45 minutes (adjust time based on age and extent of calcification e.g. if Apoe w/ 16wk HFD incubate for 45 minutes). At the end of the incubation, mince thoroughly with scissors for a few minutes (<5min) until tissue is mostly dissolved. 7) Pipet up and down ~20-30 times to thoroughly disrupt tissue. Confirm full digestion under the microscope (single cells w/o clumps). If the tissue is not fully digested after pipetting incubate at 37C for an additional 10-15 minutes (may lead to increased cell lysis). 8) Transfer cell suspension into 1.5mL Eppendorf and centrifuge at 500xg for 5 minutes. 9) Carefully remove supernatant (confirm that the cell pellet is still present). 10) Resuspend the cells in 200uL HBSS (adjust volume as needed). Cell solution was then sorted for live cells on FACS and then proceeded to 10X capture following 10X protocol. 11) Immediately prior to FACS and/or staining, pass cells through a 35um strainer. Note: Coating collection tubes in BSA solution may improve cell survival. scRNA libraries were prepared following 10X protocol. cDNA libraries were then sequenced to at least 50K reads per cell.