GSM9814482: sorted cortical microglia, from MG-srgap2f/f mice, rep2 (mouse 1062); Mus musculus; RNA-Seq
Fresh cortices from ice-cold PBS-perfused anesthetized P30 mice were quickly homogenized made into a single-cell suspension by passing it through a 100 mm filter. Immune cells were isolated by centrifuging a brain homogenate on a 40% Percoll gradient at 2000 rpm for 5 min at 4ºC. Myelin and fat debris were removed, cells washed with PBS, and incubated (2% FBS in PBS) with the following antibodies for 30 min at 4ºC: CD11b-APC (1:200,Tonbo), CD45-APCCy7 (1:200 Tonbo), Cx3Cr1-PECy7 (1:200 Biolegend). After washing twice, microglia (~100,000 cells / cortex) were sorted directly into TRIzol (Thermo Fisher Scientific) as CD11b+, CD45low, CX3CR1+ cells on a Sony MA900 (SONY) sorter with a 100 mm microfluidic chip. TRIzol containing the cell lysates was mixed with chloroform at a 5/1 ratio, shaken and spun at 12,000 g for 15 min at 4ºC. Clear supernatant was transferred to a fresh tube and washed with 70% ethanol. RNA cleanup was performed on a RNeasy Mini (Qiagen) according to manufacturer instructions. RNA quantity and quality was assessed with a 2100 Bioanalyzer (Agilent). cDNA Libraries were prepared from samples with >8.5 RIN using the NEBNext Ultra II RNA Library Prep quit, the NEBNext Poly(A) mRNA Magnetic Isolation beads, and the NEBNex Multiplex Oligos for Illumina (all New England Biolabs), all according to manufacturer's instructions.