GSM7416851: Control.RFP; Mus musculus; RNA-Seq
For each sample, the ascending aortas of 3 mice were pooled together in ice-cold Hanks' balanced salt solution (HBSS, without Ca2+ and Mg2+) (Gibco, Waltham, MA) with 10% fetal bovine serum (FBS). Aortas were cut into small pieces with microsurgical scissors and then placed into an enzyme cocktail prepared with 3 mg/ml collagenase type II (LS004176, Worthington Biochemical Corp., Lakewood, NJ), 0.15 mg/ml collagenase type XI (H3506, Sigma Corp., Kanagawa, Japan), 0.1875 mg/ml lyophilized elastase (LS002292, Worthington), 0.24 mg/ml hyaluronidase type I (H3506, Sigma), and 2.38 mg/ml 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, H4034, Sigma) in HBSS (with Ca2+ and Mg2+) (14025092, Thermo Fisher Scientific, Waltham, MA). Tissues were digested in a 37 °C water bath for 1 to 2 hours, and dissociation was examined under a microscope. Cell suspensions were collected by using a 40-µm cell strainer (CLS431750-50EA, Corning, Inc., Corning, NY), centrifuged at 300 x g for 10 minutes, and resuspended in HBSS (without Ca2+ and Mg2+) (14175095, Thermo Fisher) with 5% FBS, followed by incubation on ice for 30 minutes. Cells were then stained by using a live and dead cell kit (L3224, Thermo Fisher) and were submitted for flow cytometry (BD Biosciences) to collect live singlet cells. The living cell rate was determined with a microscope by using trypan blue (T8154, Sigma Corp.) staining. Single-cell suspensions were separately dispensed onto the Chromium Controller (10x Genomics, Pleasanton, CA); we targeted 10,000 cells per sample. Our scRNA-seq library was constructed by using the Chromium Single-Cell 3′ v3 Reagent Kit (10X Genomics).