title: Illumina HiSeq 4000 sequencing; Closing the mitochondrial permeability transition pore in hiPSC-derived endothelial cells induces glycocalyx formation and functional maturation.
design: Closing the mitochondrial permeability transition pore in hiPSC-derived endothelial cells induces glycocalyx formation and functional maturation.
library_construction_protocol: Shear experiments were performed using an Ibidi flow system (Ibidi). Cells were cultured for 4 days at a constant laminar shear stress of 5 dyne/cm2, in EC-SFM full medium as previously described (Boels et al., 2016; van den Berg, 2019). Cells were seeded into closed perfusion chambers (ibiTreat 0.4 µ-Slide I or VI, Luer) at a concentration of 1.4*106 cells per mL and allowed to adhere for 3 hours. Thereafter, the chamber was connected to a computer-controlled air pressure pump and a fluidic unit with a two-ways switching valve. The pump setup allowed pumping of 16 mL cell culture medium from two reservoirs in a unidirectional way through the flow channel over the monolayer of ECs at a constant shear stress of 5 dyne/cm2. Medium was refreshed after 1 day of culture. The chamber and the reservoirs containing the medium are kept in an incubator at 37 °C and 5% CO2. RNA was isolated from cells subjected to shear stress in a 0.4 µ-Slide I Luer flow chamber. RNA isolation in static condition was performed after cells cultured in a 6-well plate reached a confluent state (4 days) using Trizol.  RNA was purified from the Trizol solution using a RNAeasy mini kit (Qiagen). Samples from 3 independent experiments were used for RNA sequencing. For each sample, an indexed cDNA library was prepared from 1µg total RNA using the KAPA stranded mRNA-seq kit (Sopachem). Clusters were generated using the Cbot2 system (Illumina).