2025-03-02
ENA first public
ERC000011
ENA-CHECKLIST
European Institute of Oncology
INSDC center name
public
INSDC status
ERS22881942
SRA accession
E-MTAB-14744:Sample 14
Submitter Id
ArrayExpress
broker name
primary cell line
cell_line
astrocyte
cell_type
not collected
collection date
human
common name
Protocols: 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.
description
normal
disease
wild type genotype
genotype
not collected
geographic location (country and/or sea)
infection with Escherichia coli
infect
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
brain
organism part
Homo sapiens
scientific_name
Sample 14
title