title: NextSeq 500 sequencing; Transcriptome analysis of 3D model of blood-brain barrier  exposed to Borrelia garini
design: Transcriptome analysis of 3D model of blood-brain barrier  exposed to Borrelia garini
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 Borrelia garini 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.   B. garini was cultured in commercially prepared BSK medium especially for Borrelia at 30°C. 3D spheroids (model of blood-brain barrier) were cultured in 24-well plate (ultra-low attachment surface) and were incubated with live Borrelia garini (MOI 0.5), bacterial cells were washed with minimal essential medium, Sigma). 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).