characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-02-20T00:15:54Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-02-20T00:15:54Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA10792381
characteristics.characteristic: External Id
characteristics.text: Division of Infection & Immunity, UCL
characteristics.characteristic: INSDC center name
characteristics.text: 2022-02-20T00:15:54Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-02-20T00:15:54Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS8443613
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11129:HA6_T45_2_S3
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: hCMEC/D3
characteristics.characteristic: cell line
characteristics.text: immortal cell line cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CLO_0000019
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: For the monoculture cell transcriptome experiments, the cells were cultivated in 6 well plates and stimulated for 4 hours on day 6. The supernatants were then removed, and the stimulated cells lysed with 600μl of buffer RLT (Qiagen). This was stored at -80°C until extraction.For the cocultured cells, the surface of basal chambers of 6 well transwell plates (Corning) were coated with 400ul of 15μg/ml poly-l-lysine overnight, then washed with PBS. HBVP were seeded onto the surface of the basal chambers at 5000/cm2 and allowed to adhere for 24 hours. 6.5mm diameter polycarbonate membrane transwells with 3μm pores were coated with 100μl of 75μg/ml rat collagen-I solution for 2 hours, then washed with PBS. The HBVP seeding solution was then replaced with 400ul of complete EBM-2 in each basal chamber and hCMEC/D3 cells were seeded at 50 000/cm2 onto the apical surface of the transwell membrane. The coculture transwells were stimulated for 4 hours on day 6. The transwells were then moved into a new 6 well plates and supernatants were removed from both transwells and basal chambers. The cells were then lysed separately, each with 600μl of buffer RLT (Qiagen). This was stored at -80°C until extraction. RNA samples in buffer RLT were thawed and extracted using RNeasy Mini Spin Columns according to the manufacturer’s instructions. RNA was eluted in 30μl of molecular grade water (Sigma) and quantified using a Nanodrop 2000 spectrometer.Contaminating DNA was removed through treatment with Turbo DNA-Free (Thermo), as per the manufacturer’s instructions RNA samples were provided to the PGU facility at UCL for library preparation and RNA sequencing using the Kappa Hyperprep kit (Roche) with polyA selection of mRNA and fragment size of 300-350 base pairs. During this process a unique index adapter sequence is added to each sample to enable multiplexing and then identification and sorting of libraries during computational analysis.
characteristics.characteristic: description
characteristics.text: monoculture
characteristics.characteristic: growth condition
characteristics.text: Streptococcus pneumoniae
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_1313
characteristics.characteristic: infect
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: vasculature of brain
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: HA6_T45_2_S3
characteristics.characteristic: title