K6NAL500
mixed sample
Protocols: Rat microglia: to prepare rat microglia, on experimental day 1 10-12 wild-type Sprague-Dawley rat pups (mixed gender) of postnatal day 0-2 were decapitated. Their cortices were then dissected under a microscope and dissociated first chemically with papain solution and then mechanically with 5 ml pipettes. The cell suspension was then plated down in DMEM medium in poly-D-lysine coated T75 plates and grown at 37°C for 10-14 days. Human astrocytes: On day 2, primary human astrocytes (purchased from Caltag Medsystems, catalogue no. SC1800, which had been isolated from the cerebral cortex, and were negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi) were recovered from liquid nitrogen, plated down in human astrocyte medium first in a T75 flask and then split into 24-well plates the next day. They were then left to grow for 1-3 days until 80-90% confluency. Mouse neurons: On day 4 or 5, wild type CD1 mouse embryos (mixed gender) of day 17.5 were decapitated and cortices were dissected and dissociated as above and 9-13 X 104 cells (in NBA medium with 1% serum) per well were plated down on top of the human astrocytes prepared earlier. The neuron-astrocyte co-culture was left to grow for a further 3-4 days. On day 11 or 12, the rat microglia were harvested by shaking in an orbital shaker and plated down in NBA medium without serum on top of either neurons, astrocytes, or the neuron-astrocyte co-culture and were left to grow for 3 days. After 3 days of co-culture, the medium was replaced with Tmo for two hours. 500 ng LPS was then added to the cells for 12-16 hours. RNA was harvested using a Roche RNA isolation Kit according to standard protocol and the quality of total RNA (RIN) was confirmed on RNA 6000 Nano Chips and an Agilent 2100 Bioanalyzer following the manufacturer's instructions. RNA-seq libraries were prepared by Edinburgh Genomics using the Illumina TruSeq stranded mRNA-seq kit, according to the manufacturer's protocol (Illumina).
ENA first public
2018-08-09
ENA last update
2017-08-22
External Id
SAMEA104223512
INSDC center alias
Centre for Integrative Physiology, University of Edinburgh
INSDC center name
Centre for Integrative Physiology, University of Edinburgh
INSDC first public
2018-08-09T17:03:13Z
INSDC last update
2017-08-22T14:57:16Z
INSDC status
public
Submitter Id
E-MTAB-5987:K6NAL500
broker name
ArrayExpress
growth condition
co-culture between mouse neurons and human astrocytes
sample name
E-MTAB-5987:K6NAL500