GSM3453852: twoN_CPC; Mus musculus; RNA-Seq
Adult c-Kit+ CPCs were isolated and expanded as previously described. Twelve-week-old FVB female mice were heparinized and anesthetized by ketamine-xylazine solution. Hearts were removed from the chest cavity and perfused on a Langendorff system at 1mL/min flow rate at 37ºC with perfusion buffer (NaCl, KCl, KH2PO4, Na2HPO4, MgSO4•7H2O, NaHCO3, KHCO3 HEPES, Taurine, Glucose, and BDM) for blood removal, and tissue was subsequently digested for 10-15 minutes with Liberase DH digestion buffer (Roche 05401089001, 5 mg/mL in perfusion buffer). Heart was collected in STOP buffer (10% Bovine calf serum, 12.5 µM CaCl2), minced into 1 mm3 and dissociated through pipetting. The non-myocyte population was separated through serial cell straining (100µm, 40µm, and 30µm pore size), followed by centrifugation at ~300 rfc for 10 minutes. Lin-cKit+ CPCs were obtained by immunomagnetic sorting with Lineage depletion kit and CD117-conjugated Microbeads (Miltenyi Biotech 130-048-102). Fresh isolated CPCs were separated for immediate single-cell RNA-Seq analysis or cultured in growth medium ((Dulbecco's modified Eagle's medium, 10% embryonic stem cells FBS, 1% insulin-transferrin-selenium, leukemia inhibitory factor [10 ng/ml], basic fibroblast growth factor [10 ng/ml], epidermal growth factor [20 ng/ml], L-glutamine [0.07mg/mL], 1% penicillin-streptomycin-glutamine, 0.1% gentamicin, 2% B27 supplement, 1% N2 supplement). Cells were plated on a pre-coated (0.1% porcine gelatin) cell culture-grade dish. Adherent cultures were expanded and passaged five times when 40% confluency was reached and used for single-cell RNA-seq. The Chromium Single Cell 3' Library & Gel Bead Kit v2 (PN- 120237), Chromium Single Cell 3' Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturers instructions in the Chromium Single Cell 3' Reagents Kits V2 User Guide.
GEO
GSM3453852