Illumina HiSeq 2500 paired end sequencing; Transcriptome analysis of HUVEC
Transcriptome analysis of HUVEC
Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords from four different donors, as previously described (Cooke, B.M., Usami, S., Perry, I., and Nash, G.B. (1993). A Simplified Method for Culture of Endothelial-Cells and Analysis of Adhesion of Blood-Cells under Conditions of Flow. Microvasc Res 45, 33-45)  Cells were maintained in Medium 199 (M199, Invitrogen) containing 20% fetal calf serum, 28μg/ml gentamycin, 2.5μg/ml amphotericin B, 1ng/ml epidermal growth factor and 1μg/ml hydrocortisone (all from Sigma) for 48 hours prior to processing. HUVEC cultures isolated using this method were 96-98% pure, determined by positive staining by flow cytometry of CD105, CD31 and vWF and the expression of elevated levels of intracellular adhesion molecule (ICAM-1) and E-selectin following stimulation with the inflammatory cytokine interleukin-1β.  Total HUVEC RNA was isolated using the RNeasy mini kit with QIAshredder (Qiagen) according to the manufacturer’s instructions. RNA integrity number was >8.0 for all samples.  Libraries were prepared using Illumina TruSeq RNA Library Preparation Kit v2, with poly-A enrichment