title: NextSeq 500 sequencing; Transcriptome analysis of human brain microvascular endothelial cells response to Streptococcus pneumoniae and its antigen Adhesion lipoprotein using RNA-seq
design: Transcriptome analysis of human brain microvascular endothelial cells response to Streptococcus pneumoniae and its antigen Adhesion lipoprotein using RNA-seq
library_construction_protocol: Human brain microvascular endothelial cells (hBMECs) were cultured in 6-well plates then incubated either with live Streptococcus pneumoniae or recombinant Adhesion lipoprotein 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.  S. pneumoniae (SP) was plated on Columbia agar blood base containing 5% sheep blood and a single isolated colony was transferred into 100 mL of Todd Hewitt Broth. Bacteria were grown at 37°C and 5% CO2 until OD600= 0.4 (mid-exponential phase).   Human brain endothelial cells cultured in 6-well plates were incubated either with live Streptococcus pneumoniae (MOI 0.5, bacterial cells were washed with minimal essential medium, Sigma) or recombinant Adhesion lipoprotein (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: infect
attributes.value: none
attributes.tag: Experimental Factor: stimulus
attributes.value: none
attributes.tag: Experimental Factor: time
attributes.value: 0