GSM6212675: P4; Mus musculus; RNA-Seq
For isolation of cells from neonatal mice, mice were sacrificed, and hearts were transferred to PBS containing heparin sodium (1000 units in 50 ml PBS) for washing. Ventricles were chopped into small pieces, and digested with the Pierce™ Primary Cardiomyocyte Isolation Kit (Thermo, 88281) according to the manufacturer's instructions with minor modifications. To obtain relatively pure cardiomyocytes and non-cardiomyocytes, we used the differential velocity adherence method to culture isolated cells for 1 hour, and non-adherent cells were collected as cardiomyocytes. For isolation of cells from adult mouse hearts, mice were injected with 100 μl heparin sodium (1000 units in 50 ml PBS) 20 minutes before sacrifice, and hearts were transferred to Tyrode's solution for washing, after which they underwent digestion using the Langendorff perfusion technique. In brief, hearts were perfused with Tyrode's solution for 5 min using the Langendorff apparatus, followed by digestion with the heart dissociation enzyme stock solution (0.7 mg/ml Collagenase type II and 0.7 mg/ml Bovine Serum Albumin Tyrode's Solution) for 30 minutes. Tissues from ventricles were collected, chopped into pieces, and gently dissociated into single cells. Digestion was stopped by Tyrode's solution containing 10% FBS. Finally, cell suspension was centrifuged (100 × g, 2 min at room temperature) three times to separate cardiomyocytes from non-cardiomyocytes. scRNA-seq libraries were generated using the ICELL8 single-cell system (Takara Bio). Briefly, isolated cells were stained with a mixture of Hoechst 33342 and Propidium Iodide (PI) (Thermo, R37610) per manufacturer's instructions. The cells were counted on the MoxiTM Z Automated Cell Counter after PBS wash. A cell suspension of 20,000 cells/ml was transferred to the SmartChipTM MultiSample NanoDispenser (MSND, Takara Bio) for single cell preparation. Dispensed cells were then imaged on the imaging station, and live single cells were selected based on Hoechst+PI- staining. The reverse transcription and first-step amplification were performed on a Chip Cycler (Bio-Rad), followed by cDNA purification and size-selection with Agencourt AMPure XP beads (A63880, Beckman Coulter). One nanogram of purified cDNA was used to generate a sequencing library with the Nextera XT DNA Library Prep Kit (Illumina, FC-131-1024), and then sequenced on the NextSeq 500 sequencer (Illumina) using the 26nt and 50nt paired-end sequencing protocol.