GSM8381209: Microglia-Cortex, Saline, 3days, Control Saline Sample A3; Mus musculus; RNA-Seq
For adult mouse brains, the Adult Brain Dissociation Kit (Miltenyi Biotec), was used. After myelin removal, total cells were counted to proceed to the MACS step. Cell suspensions containing approximately 1x10E6 cells were labeled with superparamagnetic MicroBeads coupled to antibodies specific for the astrocyte marker ACSA-2 (Anti-ACSA-2 MicroBead Kit, mouse; Miltenyi Biotec). Before antibody labeling, nonspecific binding to the Fc receptor was blocked using the FcR Blocking Reagent, mouse (Miltenyi Biotec). Cells were suspended in PBS with 0.5% BSA and the cell suspension was loaded onto an MS Column (Miltenyi Biotec), which was placed in the magnetic field of a MiniMACSTM Separator (Miltenyi Biotec). The magnetically labeled ACSA-2-positive cells were retained within the column and eluted as the positively selected cell fraction after removing the column from the magnet. Subsequently, the negatively selected fraction was incubated with Anti-CD11b Magnetic Microbeads (130–093-634, Miltenyi Biotec) in PBS with 0.5% BSA. The cell suspension was loaded onto an MS Column (Miltenyi Biotec), which was placed in the magnetic field of a MiniMACSTM Separator (Miltenyi Biotec). The magnetically labeled CD11b-positive cells were retained within the column and eluted as the positively selected cell fraction after removing the column from the magnet Library was constructed using the Illumina Stranded mRNA prep, according to the manufacturers instructions