Illumina NovaSeq 6000 sequencing; Single-cell RNA-sequencing of cardiac cells isolated from infant mouse at postnatal 3 weeks
Single-cell RNA-sequencing of cardiac cells isolated from infant mouse at postnatal 3 weeks
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. There are no nucleic acid extraction steps using the 10X Chromium Single Cell Platform (10X Genomics) according to the manufacturer's protocol (Single Cell 3′ library and Gel Bead Kit V3). Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 1,045 cells per μl (Single Cell 3′ library and Gel Bead Kit V3, 4 rxns PN-1000092) as described in the manufacturer's protocol. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer's protocol.
Experimental Factor: organism part
heart