Endothelial Cell Culture and RNA Isolation

Cell cultures were performed according to the protocol described in detail in our previous work [26 (link)]. Briefly, we used iCell Endothelial Cells (Cellular Dynamics, Madison, WI, USA), which are purified human endothelial cells differentiated from induced pluripotent stem cells, cultured in Vasculite Maintenance Medium (Cellular Dynamics) according to the manufacturer’s protocol. After obtaining confluence of 70–85%, total RNA was isolated using silica-based minicolumns according to the manufacturer’s protocol (TotalRNA Mini, A&A Biotechnology, Gdansk, Poland). The concentration and purity of the RNA eluent were measured by spectrophotometry (NanoDrop Spectrophotometer 2000, NanoDrop Technologies, Wilmington, DE, USA). The quality and integrity of RNA were assessed with Bioanalyzer (PicoRNA Chip, Agilent Technologies, Santa Clara, CA, USA). The resulting RNA was used to prepare libraries preceded by ribodepletion according to the standard Illumina protocol.