BGISEQ-500 paired end sequencing; Endothelial cell heterogeneity in pathological angiogenesis characterized by metabolic transcriptome diversity
Endothelial cell heterogeneity in pathological angiogenesis characterized by metabolic transcriptome diversity
Human umbilical vein endothelial cells (HUVECs) were freshly isolated from umbilical cords obtained from multiple donors (with approval from the Medical Ethical Committee KU Leuven / UZ Leuven and informed consent obtained from all subjects) as previously described (Schoors et al., 2015), and were regularly tested for mycoplasma. The ECs were maintained on 0.1% gelatin-coated dishes in M199 medium (1 mg/ml D-glucose) (Thermo Fisher Scientific) supplemented with 20% fetal bovine serum (FBS) (Merck-Biochrom), 2 mM L-glutamine (Thermo Fisher Scientific), Endothelial Cell Growth Supplement (ECGS)/ Heparin (PromoCell), 100 IU/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific) or in endothelial cell basal medium (EGM2) (PromoCell) supplemented with endothelial cell growth medium supplement pack (PromoCell). In all experiments, HUVECs were always used as single-donor cultures and were used between passage (p) 1 and 4. To silence the expression of ALDH18A1 or SQLE, gene-specific oligonucleotides were cloned into the pLKO-shRNA2 vector (Clontech). All constructs were sequence verified. Lentiviral particles were produced in 293T cells and a MOI of 20-25 was used for lentiviral transduction. Transductions were performed on day 0 in the evening, cells were refed with fresh medium on day 1 in the morning and cells were harvested from day 3 or 4 onwards. Knockdown efficiency was monitored for each experiment either at the mRNA (qRT-PCR) or protein level or both and compared to expression in cells transduced with a negative control shRNA. Control (pLKO-transduced), ALDH18A1 and SQLE silenced cells were collected to perform RNA-seq. RNA was extracted and purified with the PureLink RNA Mini Kit (Thermo Fisher Scientific).  RNA libraries were created using the KAPA mRNA stranded library prep kit according to the manufacturer's instructions.
Experimental Factor: RNA interference
control shRNA