characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-05-02T17:01:54Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-02-24T17:16:23Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA100650418
characteristics.characteristic: External Id
characteristics.text: Centre for Integrative Physiology, University of Edinburgh
characteristics.characteristic: INSDC center name
characteristics.text: 2017-05-02T17:01:54Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-02-24T17:16:23Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1572315
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5514:3F5
characteristics.characteristic: Submitter Id
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: astrocyte species
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: mixed astrocyte and neuron
characteristics.characteristic: cell type
characteristics.text: Protocols: Astrocytes and neurons were cultured from E17.5 CD1 mouse and E20.5 Sprague Dawley rat embryos. Cortices were dissected, enzymatically digested with papain and mechanically dissociated using a 2 mL pipette. Mouse astrocytes were obtained by growing cells at low density in DMEM containing 10% serum and were passaged twice, using Trypsin (both Life Technologies), before co-culturing them with neurons in a 24-well plate. These astrocytes are >99% GFAP positive and <0.1% NeuN positive. For mixed-species co-cultures, rat neurons were plated on top of a confluent layer of DIV14 mouse astrocytes and both astrocyte mono-cultures and astrocyte-neuron co-cultures were subsequently kept in Neurobasal-A medium containing B27 (both Life Technologies), but devoid of serum. Where used, pure neuronal cultures were generated from E17.5 CD1 mouse embryos, with the anti-mitotic AraC added on the day of plating which restricts astrocyte numbers to <0.1%. All cultures were used 8-10 days post neuron plate down. Cells were transferred into media containing 10% Minimum Essential Media (MEM, Life Technologies) and 90% Salt-Glucose-Glycine (SGG) medium containing 114 mM NaCl, 0.219 % NaHCO3, 5.292 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 1 mM Glycine, 30 mM Glucose, 0.5 mM sodium pyruvate, 0.1 % Phenol Red; osmolarity 325 mosm/l) either over night or for 4h prior to stimulations. To investigate the effect of synaptic activity on astrocytic gene expression, co-cultures were transferred into TTX-containing medium on DIV8 for 22h to inhibit neuronal action potential firing. Subsequently, cells were washed with medium free of TTX and then kept in medium either containing Bicuculline (BiC, 50 μM, Tocris) to induce network bursting, or BiC and TBOA (DL-threo-β-Benzyloxyaspartic acid, 50 μM, Tocris), to increase the half-time of glutamate uptake, for 16h, after which RNA was collected. Control conditions were washed with TTX and left in medium containing either BiC or BiC/TBOA in the presence of TTX, or TTX alone. Barcoded RNA-seq libraries were prepared by Edinburgh Genomics using the Illumina TruSeq stranded mRNA-seq kit, according to the manufacturer’s protocol (Illumina).
characteristics.characteristic: description
characteristics.text: co-culture
characteristics.characteristic: growth condition
characteristics.text: Rattus norvegicus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10116
characteristics.characteristic: neuron species
characteristics.text: mixed sample
characteristics.characteristic: organism
characteristics.text: mixed sample
characteristics.characteristic: organism
characteristics.text: mixed sample
characteristics.characteristic: scientific_name
characteristics.text: 3F5
characteristics.characteristic: title