title: NextSeq 500 sequencing; Transcriptome analysis of human brain microvascular endothelial cells response to Neisseria meningitidis and its antigen MafA using RNA-seq
design: Transcriptome analysis of human brain microvascular endothelial cells response to Neisseria meningitidis and its antigen MafA using RNA-seq
library_construction_protocol: Human brain microvascular endothelial cells (hBMECs) were cultured in 6-well plates then incubated either with live N. meningitidis or recombinant MafA  or culture medium only (non-induced control) for 6 hours.  Human BMEC (hBMEC/D3 cell line), was acquired from Merck/Millipore (Prague, Czech Republic). Cells were cultured in 6-well cell culture plates covered with collagen type I (Sigma, USA) and endothelial cell growth basal medium (EBM-2; Lonza,UK; Appendix 1). Cells were incubated at 37°C in a humid atmosphere in the presence of 5% CO2.  N. meningitidis (NM, isolate M1/03, serogroup B, strain MC58).) was plated on commercially prepared Thayer Martin agar and a single isolated colony was transferred into 50 mL of Brain Heart Infusion Broth enriched with 10 mM MgCl2 . Bacteria were grown at 37°C and 5% CO2 until OD600= 0.6 (mid-exponential phase).   Human brain endothelial cells cultured in 6-well plates were incubated either with live Neisseria meningitidis (MOI 0.5,bacterial cells were washed with minimal essential medium, Sigma) or recombinant MafA (27 μg/well). Nontreated cells were used as negative control, for 6 hours at  37°C in the presence of 5% CO2. mRNA from human brain microvascular endothelial cells 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).
attributes.tag: Experimental Factor: stimulus
attributes.value: Neisserial ligand MafA