GSM3411074: SS2_16_366_J9; Mus musculus; RNA-Seq
After transcardial perfusion with PBS, brains were roughly minced and homogenized with a potter in HBSS containing 15 mM HEPES buffer and 0.54 % Glucose. Whole-brain homogenate was separated by 70/37/30 % layered Percoll gradient centrifugation at 800 g for 30 min at 4 °C (no brake). The CNS macrophages containing interphase was then collected and washed once with PBS containing 2% FCS and 10mM EDTA before staining. Cells were stained with primary antibodies directed against CD11b (M1/70, BioLegend), CD45 (30-F11, BD Biosciences), Ly6C (AL-21, BD Biosciences) and Ly6G (1A8, BD Biosciences) for 20 min, and CD206 (C068C2, BioLegend) for 45 min at 4 °C. After washing, cells were analyd using a MoFlo Astrios (Beckman Coulter). microglia were FACS-sorted from six different CNS regions of healthy and diseased brains (see gating strategy shown in supplementary Fig.2) into a 384-well plate containing a lysis buffer. Single cell RNAseq libraries were generated following the published SmartSeq2 protocol (Picelli et al. Nature Metods 2013)
GEO
GSM3411074