Microglia single-cell suspensions were generated from the brain of healthy or EAE mice (day 16 after induction) as described above. Surface staining was performed in PBS containing BioLegend CD45 (I3/2.3), CD11b (M1/70), CX3CR1 (SA011F11) and P2RY12 (S16007D) antibodies for 30 min at 4 °C. The antibody cocktail was supplemented with hashtag antibodies (TotalSeq-B0301/B0302/B0303/B0304 anti-mouse hashtag 1/2/3/4, Biolegend) to label individual animals. Microglia were isolated by fluorescence-activated cell sorting (FACS) for the presence of CD45 (intermediate), CD11b, CX3CR1 and P2RY12 signals. Cells were sorted from individual animals, washed, counted and concentrated to 1000 cells/µl before pooling. Four independent pools were generated, each containing equal numbers of cells from healthy CD83wtCre, healthy CD83∆MG, EAE CD83wtCre and EAE CD83∆MG animals (16 animals in total, 4 biological replicates per condition in 4 pools, 1 pool containing all 4 conditions). Up to 25,000 cells (hyper-loading) pooled from four animals were loaded into a single well of a Chromium chip G (10x Genomics). 3’ gene expression libraries were generated using Chromium Next GEM Single Cell 3’ Kit 3.1 with 3’ Feature Barcode Kit and dual indexing
