RNA-seq transcriptome profiling of human pulmonary artery endothelial cells and brain microvascular endothelal cells in response to hypoxia

In this study, we analzyed differences in the effect of hypoxia on the transcriptomic profile of cultured human pulmonary artery endothelial cells (HPAECs) vs. human brain microvascular endothelial cells (HBMVECs). To determine which hypoxia-regulated genes were dependent on hypoxia inducible factor 1-alpha (HIF-1alpha), some cells were transfected with an siRNA against HIF-1alpha prior to treatment with hypoxia. At the completion of the treatment protocol, mRNA was isolated using standard methodology and analyzed further by RNA-seq. Control human pulmonary artery endothelial cells (HPAECs) or human brain microvascular endothelal cells (HBMVECs) from male and female donors were grown under standard tissue culture conditions. Confluent cells (N=3/condition) were treated with normoxia (21% O2) or hypoxia (0.2% O2) for 24 hr. An additional group of HPAECs and HBMVECs were transfected with siRNA-HIF-1A prior to treatment with hypoxia. At the completion of the treatment protocol, mRNA was isolated and subjected to RNA-seq analysis. The Total-RNA samples were quantified using an Agilent 2100 Bioanalyzer instrument, with a corresponding Agilent Bioanalyzer RNA Nano assay. The resulting RIN (RNA Integrity Number) scoresand concentrations were considered for qualifying samples to proceed. Sequencing was performeed using an Illumina NextSeq 500 instrument, with a High-Output 150-cycle kit to obtain Paired-End 75bp reads. The pool was loaded at 1.9pM, with 5% PhiX spikedmin as a sequencing control.