GSM8717594: set3_12_6_2023; Mus musculus; RNA-Seq
Aortic arches were isolated from all groups after saline perfusion via cardiac puncture. Minced tissue was suspended in digestion buffer containing liberase (273582, Roche, Switzerland), Hyaluronidase (3506, Sigma Aldrich, MO) and DNase I (DN25, Sigma Aldrich, MO) in 1% BSA- Hank's Balanced Salt Solution and 1mM EDTA. Tissue was incubated at 37oC for 15 minutes using C-tubes, placed in genlteMACS dissociator (Miltenyi Biotech, Germany), as previously described (30). The digested tissue was filtered with a 100μm cell strainer, washed with 1X cold PBS and centrifuged at 350g for 5 minutes at 4oC. Aortic arch cells were resuspended in eBioscience™ Fixable Viability Dye eFluor™ 660 (65-0864-14, ThermoFisher, MA) for dead cell staining, and in PE/Cyanine7 anti-mouse CD45 Antibody (103113, Biolegend, CA). Alive CD45+ cells were sorted in fluorescent-activated cell sorter (FACS) SY3200 highly automated parallel sorting (HAPS) (100μm) cytometer, for downstream single cell RNA sequencing. Isolated cells were loaded into single-cell gel beads and barcoded with a unique molecular identifier using the Single cell 3' reagent kit (10X Genomics, 3' CellPlex Kit Set A and Cell Multiplexing Oligos; CMO). For set 2, CMO308 is a baseline sample; CMO309 is regression control; CMO310 and CMO11 are regression depleted. For set 3, CMO304 is a baseline sample; CMO305 and CMO307 are regression depleted; CMO306 is regression control.