GSM8988020: Heart_Tie1_WT; Mus musculus; RNA-Seq
Hearts were minced using scissors and enzymatically dissociated in 1 mg/ml each of lyophilized collagenase type I, II and IV (Gibco) at 37°C for 25 min. The suspensions containing cell clumps were repeatedly pipetted to achieve single-cell suspension. RBC lysis buffer (Sigma-Aldrich) was used to lyze the leftover erythrocytes. Cell suspension was further filtered through the series of nylon mesh of 70, 40, 35 and 20 µm pore sizes. Single-cell suspensions were washed using DPBS with 2% heat inactivated FCS and resuspended in DPBS containing 0.04% BSA. This sample represented stromal-vascular fraction. To obtain cardiac cell suspension enriched for ECs, the cells were incubated with mouse Fc blocker (rat-anti-mouse CD16/CD32 (BD Biosciences) for 5 min followed by the antigen-specific antibody fluorescent conjugates for 30 min (list of the antibodies is in Supplemental Table 12). The live cardiac ECs (CD31+ CD45- Ter119- CD140a- DAPI-) were sorted in FACS Aria II (BD Biosciences). After the FACS step, cardiac ECs were washed using DPBS with 2% heat inactivated FCS and were resuspended in DPBS containing 0.04% of BSA. The cell viability and cell cluster levels of FACS sorted ECs were determined using automated cell counter (Luna, Logos Biosystems). The single cells were captured using 10X chromium Single-Cell 3'RNA sequencing system (10x genomics, Pleasanton, CA) with the reagent kit v2 according to the manufacturer's instructions. In essentially the same way the cell suspensions from kidney and lung were obtained by enzymatic digestion, enriched for ECs by FACS-sorting and prepared for scRNAseq analysis (PMID: 35050301). Library was performed according to the manufacturer`s instructions (single cell 3' protocol, 10x Genomics)