Notch signaling activation in hPSC-derived CNS-like endothelial cells induces differential expression of blood-brain barrier-related genes
Notch signaling activation in hPSC-derived CNS-like endothelial cells induces differential expression of blood-brain barrier-related genes
hPSC-CECs treated with doxycycline (Dox) or PBS were sorted into subpopulations by FACS based on level of surface CD144 expression (CD144hi and CD144lo). Cells were lysed with TRIzol reagent (Invitrogen) for extraction of total RNA, which was subsequently purified using the Direct-zol RNA Miniprep Kit (Zymo Research). Samples were incubated with RNase-free DNase I (Qiagen) to eliminate residual gDNA during RNA Miniprep protocol. RNA was eluted with RNase-free water and concentration was determined using the Qubit 4 Fluorometer (Invitrogen) and Qubit RNA High Sensitivity Assay Kit (Invitrogen). Purified RNA samples (â‰¥200 ng) were sent to Novogene Corporation (Sacramento, CA) for library preparation and mRNA sequencing.  Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.