GSM7233707: Pool3_GEX; Mus musculus; RNA-Seq
Mice were perfused with ice-cold PBS by cardiac puncture and brains were collected in RPMI medium on ice. Tissue was homogenised using a tissue dounce grinding set. Homogenates were subjected to density centrifugation (37% Percoll, 550g, 18 min) to remove myelin debris. 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 for cell sorting 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 1,000 cells/µl before pooling. Three independent pools were generated, each containing equal numbers of cells from healthy wild-type, healthy CD83 knock-out, EAE wild-type and EAE CD83 knock-out animals (12 animals in total, 3 biological replicates per condition in 3 pools, 1 pool containing all 4 conditions). Up to 25,000 cells (hyper-loading) pooled from 4 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 (10x Genomics protocol CG000316 Rev D). Libraries were sequenced as PE150 by Illumina sequencing to 65-80% saturation by the external provider Novogene.