organism: Rattus norvegicus
description: Protocols: Primary rat microglia were generated from postnatal rat pups (P0-P2). Briefly, neocortices were dissected, enzymatically digested, homogenized to single cell suspension and plated down onto poly-D-Lysine-coated flasks. Cultures were maintained for 12 days in DMEM+10% FBS, with medium replacements every 3-4 days. After this period of time, the culture contains predominantly astrocytes and microglia, since the neurons have largely died off under the sub-optimal (for them) culture conditions. To detach the weakly adherent microglia, flasks were placed on an orbital shaker shake at 250 rpm for 1 h at 37C, centrifuged to obtain a pellet, then resuspended in Neurobasal A medium and counted using a hemocytometer, adjusting the concentration to 2 X 105 cells/ml, ready for seeding onto neuron-astrocyte co-cultures or onto poly-D-Lysine-coated 24 well plates for generating mono-cultures. Human astrocytes (Caltag Medsystems cat. no. SC-1800) were maintained on poly-D-lysine-coated tissue culture flasks in Astrocyte Medium (Caltag Medsystems cat. no. SC-1801), and passaged according manufacturer's instructions. Astrocytes were always used at 7 passages or less and seeded at 3-5 X 105 cells per well of a 24-well plate (poly-D-Lysine-coated). The cells were left until 80-90% confluent, which typically took 3 days. At this point, primary mouse neocortical neurons were prepared from E17.5 CD1 mice and seeded at 1 X 105 cells per well onto the astrocyte monolayer. These co-cultures were maintained for 7 days in Neurobasal A medium, plus 1% rat serum, in the presence of AraC to prevent any mouse astrocyte proliferation, and any further human astrocyte proliferation. At this point, microglia were seeded onto the co-culture at 2 X 105 cells per well and maintained for 3 further days in Neurobasal A medium. Cells were then physically dissociated from the culture dishes in ice-cold dPBS with sodium pyruvate (ThermoFisher) + 0.4% BSA and stained with PE conjugated anti-mouse CD11b (Biolegend). A pure single-cell suspension of microglia was sorted using a BD Aria FACS system. Suspension sufficient to generate 5000 single cell GEMs (gel beads in emulsion) per sample was loaded into a Chromium controller (10X Genomics) in a Single Cell 3' Chip. Libraries were prepared using the Chromium Single Cell 3' GEM, Library and Gel Bead Kit v3 (10X Genomics) as per manufacturer's instructions.
attributes.tag: ENA first public
attributes.value: 2021-03-23
attributes.tag: ENA last update
attributes.value: 2021-01-20
attributes.tag: External Id
attributes.value: SAMEA7845255
attributes.tag: INSDC center alias
attributes.value: Centre for Discovery Brain Sciences, University of Edinburgh
attributes.tag: INSDC center name
attributes.value: Centre for Discovery Brain Sciences, University of Edinburgh
attributes.tag: INSDC first public
attributes.value: 2021-03-23T16:08:16Z
attributes.tag: INSDC last update
attributes.value: 2021-01-20T18:36:34Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10030:Sample 1
attributes.tag: age
attributes.value: 0 to 2
attributes.tag: biological sample
attributes.value: 1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: microglial cell
attributes.tag: common name
attributes.value: Norway rat
attributes.tag: culture batch
attributes.value: 1
attributes.tag: developmental stage
attributes.value: postnatal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: monoculture
attributes.tag: individual
attributes.value: pooled
attributes.tag: organism part
attributes.value: neocortex
attributes.tag: sample name
attributes.value: E-MTAB-10030:Sample 1
attributes.tag: sequencing batch
attributes.value: 1
attributes.tag: sex
attributes.value: mixed
attributes.tag: strain
attributes.value: Sprague-Dawley