title: Comparative study of gene expression in human primary astrocytes induced with 6 different neuroinvasive bacteria
design: Comparative study of gene expression in human primary astrocytes induced with 6 different neuroinvasive bacteria
library_construction_protocol: Primary human astrocytes (ScienCell, USA) were cultured in poly-L-Lysine-coated tissue culture flasks (Sigma-Aldrich, USA) with complete astrocyte medium (ScienCell) containing 2% fetal bovine serum and 1% astrocyte growth supplement. After reaching > 80% confluence, the cells were detached with trypsin EDTA, washed with astrocyte medium, and seeded in a 6-well culture plate coated with poly-L-Lysine. Cells were cultured in complete astrocyte medium at 37 °C with 5% CO2 until 90% confluence. Further experiments were performed on a confluent monolayer of approximately 300 000 cells per well.     Bacteria cultures: Six neuroinvasive bacterial strains were cultivated. Borrelia bavariensis (Borrelia), strain SKT7.1, was cultivated in BSK-II medium enriched with 6% rabbit serum (Biowest, France) at 33 °C till log phase.  Escherichia coli K1 (E. coli, neuroinvasive strain ATCC 700973) and Klebsiella pneumoniae (Klebsiella, neuroinvasive strain ATCC), were grown on lysogeny agar (Sigma-Aldrich) at 37 °C. Listeria monocytogenes (Listeria), isolated from liquor of patient admitted to The University Hospital Olomouc, Czech Republic (REF) was plated on PALCAM agar base (Merck Milipore, USA) enriched with selective supplements (OXOID, Ireland) at 37 °C. Neisseria meningitidis, (isolate M1/03, capsule deficient strain sequenced with WGS) was grown on Thyer-Martin agar (Milipore Sigma, Canada).  Streptococcus pneumoniae was grown on chocolate agar with 5% of sheep blood.  Challenge of astrocytes with Bacteria: Astrocytes were incubated with each pathogen separately (MOI - Borrelia 0.5, E. coli 0.00005, Klebsiella 0.00005, Listeria 0.032, Neisseria 0.5, or Streptococcus 0.5) at 37 °C in a 5% CO2 atmosphere for 6 hrs. Astrocytes without bacteria served as negative control. Experiment was conducted in three biological replicates.  RNA was isolated using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. RNA was treated with DNase I (Qiagen) during the isolation process. RNA concentration and purity were measured at 260/280 and 260/230 (Nanodrop, Thermo Fisher Scientific, USA). Samples were stored in aliquots at -80 °C.  Quantseq FWD kit (Lexogen, Austria) was used to prepare libraries for NGS. Reverse transcription of RNA, amplification of libraries, quality control of libraries and purification were performed exactly as described in manual provided with kit.