Illumina HiSeq 3000 sequencing; LPS response of cardiovascular endothelial cells transduced with an APEX1(1-20) expressing vector or an empty vector
LPS response of cardiovascular endothelial cells transduced with an APEX1(1-20) expressing vector or an empty vector
Primary human endothelial cells (EC) were obtained from LONZA (Cologne, Germany) and cultured as previously described [7]. In detail, EC were cultured in endothelial basal medium supplemented with 1 μg/mL hydrocortisone, 12 μg/mL bovine brain extract, 50 μg/mL gentamicin, 50 ng/mL amphotericin B, 10 ng/mL epidermal growth factor (LONZA, Cologne, Germany) and 10% fetal bovine serum until the third passage. After detachment with trypsin, cells were grown for at least 20 h. All experiments were performed with EC in passage 3. HEK293 were cultured in DMEM GlutaMAX™ supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin and then used for the production of lentiviruses. Generation of VSV-G pseudotyped lentiviral particles and transduction of EC were performed as previously described [8]. Lentiviral titers were determined with the QuickTiter Lentivirus Titer Kit (Lentivirus-Associated HIV p24) (Biocat, Heidelberg, Germany). EC were transduced with a multiplicity of infection of approximately 20. The day after transduction the cells were washed three times, the medium replaced, and cells cultivated for another day before they were treated with 150 ng/mL LPS for 18 h. Cells were lysed using TRIzol® (Thermo Fisher Scientific, Dreieich, Germany) and RNA was isolated according to the manufacturer's instructions. RNAs were subjected to a second purification step using the RNeasy® Mini kit (Qiagen, Hilden, Germany). RNA concentrations were measured using a NanoDrop™ 2000c (Thermo Fisher Scientific, Dreieich, Germany), and RNA integrity and purity were determined by agarose gel electrophoresis. RNA sequencing data were obtained from quadruplicate total RNA samples. Total RNAs used for transcriptome analyses were quantified using the Qubit RNA HS Assay kit (Thermo Fisher Scientific, Dreieich, Germany) and quality was determined by capillary electrophoresis using the FragmentAnalyzer and the Total RNA Standard Sensitivity Assay (Agilent Technologies, Santa Clara, CA, USA). All samples in this study showed highest RNA Quality Numbers (RQN 10.0). Library construction and sequencing were performed at the Genomics and Transcriptomics Laboratory at the Biological Medical Research Centre (BMFZ) of the Heinrich-Heine University Düsseldorf. Library preparation was performed according to the manufacturer's protocol using the TruSeq Stranded mRNA Assay kit (Illumina, San Diego, CA, USA). Briefly, 500 ng total RNA was used for mRNA capturing, fragmentation, synthesis of cDNA, adapter ligation and library amplification.