GSM6615885: Heart tissue, POR knockout, N1 [Heart_POR-KO_N1]; Mus musculus; RNA-Seq
Cardiac endothelial cells were isolated from LV of ecPOR-/- and CTL mice using magnetic activated cell sorting. The remaining myocardial tissue was designated as cardiac fraction. Briefly, two LVs per genotype were pooled in Hank's balanced salt solution (HBSS, 1mL) and cut into small pieces (1 mm). Minced tissue was transferred to 50 mL Falcon tubes containing enzyme mix (10 mL Dulbecco's modified Eagle's (DMEM, Gibco Life Technologies, Carlsbad, US) containing dispase (1.2 U/mL, #39307800, Roche Diagnostics, Mannheim, Germany), collagenase II (2 mg/mL, #LS004177, Worthington, Lakewood, US), elastase (0.03 mg/mL, #2292, Worthington, Lakewood, US) and digested for 20 minutes at 37 °C. Digestion was stopped with DMEM containing 10% FCS. Undigested tissue was removed by filtration through strainers (100 µm and 70 µm). Cells were spun down (10 min, 500 xg, 4 °C) and the pellet was resuspended in 10 mL RBC (redo blood cells) lysis buffer (J62150, Alfa Aesar, Kandel, Germany) RNA and library preparation integrity were verified with LabChip Gx Touch 24 (Perkin Elmer). For the cardiac fraction 4 µg of total RNA was used as input for VAHTS Stranded mRNA-seq Library preparation following manufacture's protocol (Vazyme). For EC samples RNA was used for SMART-Seq® v4 Ultra® Low Input RNA Kit (Takara Bio).