GSM8023821: 1-month PI PBS-injected P301L, GEX; Mus musculus; RNA-Seq
After conducting PBS perfusion and removing the cerebellum and olfactory bulbs, contralateral hemispheres (i.e., opposite to the injected hemisphere) were subjected to mechanical and enzymatic dissociation using the GentleMACS™ dissociator (130-093-235, Miltenyi Biotec) in combination with the Neural Tissue Dissociation Kit (130-092-628, Miltenyi Biotec), according to the manufacturer's instructions. To avoid microglial activation during extraction, we dissociated the hemispheres in the presence of Actinomycin D (5 nM, Sigma-Aldrich SBR00013) and Brefeldin A (1x, 420601 BioLegend). After enzymatic dissociation, cells were resuspended in 30% Percoll (P1644, Sigma-Aldrich) and centrifuged for 15 min at 300 g at 4°C for myelin removal. The supernatant containing the myelin was removed, and the pelleted cells were washed with FACS buffer (PBS, 0.5 % BSA, 2 mM EDTA). Up to 2 million cells per mouse were incubated for 30 min on ice with 25µL of staining mix in PBS containing 0.04% BSA, Cluster of Differentiation CD11b-PE-Cyanine5 antibody (BioLegend, cat 101210), TruStain FcX Block (BioLegend, cat 101320) and a mouse CITE-Seq antibody panel.  The DAPI-CD11b+ cells were subsequently isolated using fluorescence-activated cell sorting (FACS) Aria II. After FACS sorting cells were spun down, the cell pellet was resuspended and incubated on ice in staining mix and the antibody panel. Replicates were pooled using hahsing antibodies. The cell suspensions were loaded on a Chromium GemCode Single Cell Instrument (10X Genomics) for GEM creation with the 3' Gel Bead and Library kit version NextGEM 3.1. according to manufacurers instructions.