RNA-seq of primary human brain endothelial cells cultured on hydrogel substrates and exposed to static or fluid shear stress conditions
RNA-seq of primary human brain endothelial cells cultured on hydrogel substrates and exposed to static or fluid shear stress conditions
Following static or FSS culture, devices were removed from perfusion. Media was aspirated, cells were kept on GEL substrates and washed with PBS and immediately incubated with TRIzol for RNA extraction. Primary human BMECs were purchased from Cell Systems (ACBRI 376) and maintained in Endothelial Cell Growth Media (C-22110, Sigma) up to passage 9. GEL substrates were coated with 50 ug/mL human collagen IV (C5533, Sigma) and 25 ug/mL bovine fibronectin (F1141, Sigma) for 24 hours before cell subculture. BMECs were subcultured on coated GELs either in glass plates (static condition) or in ibidi devices (u-Slide I Luer 3D, ibidi) for the FSS condition. All cells were subcultured at a density of 1e6 cells/mL and allowed to adhere for 24 hours before beginning FSS experiments. BMECs in ibidi devices were subjected to 1.7 dyne/cm2 FSS for 48 hours (following initial 24 hours of flow conditioning at 0.2 dyne) in endothelial cell media supplemented with 3% (w/v) 70kDa dextran and 1X penicillin-streptomycin. Static cultures were maintained in glass plates for 48 hours, with identical media supplements. At the end of each experiment, cells were lysed with TRIzol and RNA was extracted using a Direct-zol RNA miniprep kit (R2050, Zymo). Purified RNA samples were submitted to the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core for bulk sequencing. cDNA libraries were prepared with a stranded mRNA (polyA-selected) library preparation kit. 