GSM8798798: Slc2a5 KO Microglia,OGD 6 hours and recovery for 1 day,rep3; Mus musculus; RNA-Seq
Mice at an age of postnatal day (P)30–45 were anesthetized with isoflurane. They were perfused with cold PBS(Corning) through the heart. Brains were isolated and immediately immersed into cold hibernation medium (5mM KCl, 5mM NaOH, 5mM NaH2PO4, 0.5mM MgCl2, 20mM Na Pyruvate, 5.5mM Glucose, 200mM Sorbitol, pH 7.2-7.4, filtered with 0.22μm filters). Dissected brain hemispheres were cut into small pieces and incubated in cold hibernation medium (BrainBits, USA) containing papain (2mg /ml, Sigma) and DNase I (100 units, Sigma) at 37˚C for 30 min with intermittent flicking. The tissue was triturated into individual cell suspension by a siliconized Pasteur pipette with a fire-polished tip (BrainBits). Cell suspensions were added into Optiprep gradient (Brewer and Torricelli, 2007b) and centrifuged at 900 g for 15min at 4℃. We then removed most upper 5ml debris and collected specific Optiprep layers (neurons and astrocytes were enriched in upper layers while microglia were enriched in pellets). Cells from different layers were washed with cold hibernation medium without Ca2+ and Mg2+(BrainBits) and centrifuged at 600 g for 10 min at 4℃. Cell pellets were resuspended with a 500µl cold hibernation medium for sorting. The RNA sequencing library was prepared according to the protocol for the SMARTer Stranded Total RNA-Seq Kit v2.
GEO
GSM8798798_r1