title: NextSeq 500 sequencing; Transcriptome analysis of 3D model of blood-brain barrier  exposed to tick-borne encephalitis virus (TBEV)
design: Transcriptome analysis of 3D model of blood-brain barrier  exposed to tick-borne encephalitis virus (TBEV)
library_construction_protocol: Human brain microvascular endothelial cells (hBMECs, human pericytes and human astrocytes were cultured in 24-well plates (ultra-low attachment surface)until 3D spheroid formation was created and then incubated with live TBEV in 3D spheroid medium or culture medium only (non-induced control) for 24 hours.  Human BMEC (hBMEC/D3 cell line), was acquired from Merck/Millipore (Prague, Czech Republic), human astrocytes and human pericytes were obtained from ScienCell company. Cells were cultured separately on T-25 culture flasks as per manufacturer´s instructions as they reach confluent monolayer. Cells were collected and seeded on 24 well plate (ultra-low attachment surface). Cells were incubated at 37°C in a humid atmosphere in the presence of 5% CO2 in 3D medium (Sciencell) until 3D spheroids were created.   3D spheroids (model of blood-brain barrier) were cultured in 24-well plate (ultra-low attachment surface) and were incubated with TBEV (1 milion virions) for 24 hours at  37°C in the presence of 5% CO2. Nontreated cells were used as negative control,  for 24 hours at  37°C in the presence of 5% CO2. mRNA from 3D formation was isolated using RNeasy Mini Kit (Qiagen, Germany) according to manufacturer's instructions. 250 ng of RNA were reverse transcribed with oligodT primers using QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen, Austria).