Illumina HiSeq 2500 paired end sequencing; RNA-seq of mouse astrocytes in monoculture and co-cultured with rat neurons (both inactive and active).
RNA-seq of mouse astrocytes in monoculture and co-cultured with rat neurons (both inactive and active).
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).
Experimental Factor: co-culture
growth condition
Experimental Factor: BiC + TBOA
compound