Single-cell RNA-sequencing of cardiac cells isolated from infant mouse at postnatal 3 weeks

To study the gene profiling of different cardiac adipogenic progenitor populations, we isolated individual cells from postnatal 3-week heart and performed single-cell RNA-sequencing. Single cell suspensions from isolated mouse heart were prepared by retrograde Langendorff perfusion. Briefly, mouse at P3W was administered with 0.2 mL heparin sodium salt solution (1000 IU/mL) via intraperitoneal injection and then euthanized by CO2 asphyxiation. The heart was harvested and hung onto the Langendorff apparatus, followed by perfusing with modified Tyrode's solution (MTS) for 2 min. The heart was next perfused with MTS containing 0.4 mg/ mL collagenase II and 25 µg/ mL DNase I for 10 min. After removed from cannula, atria and valves were removed and ventricles were minced with fine forceps. Cells were filtered through a 70 µm cell strainer and centrifuged at 30x g for 5 minutes at 4°C to remove most cardiomyocytes. The cell suspension was carefully transferred into a new 15 mL centrifuge tube and centrifuged at 300x g for 5 minutes at 4°C. Cell pellet was washed and suspended in DMEM containing 10% FBS for single-cell sequencing. Single cell library was generated using 10x Genomic Chromium system with the v3 single cell reagent kit. Sequencing of library was performed on Illumina NovoSeq 6000 platform.