GSM6267636: H9; Mus musculus; RNA-Seq
Blood was thoroughly removed by transcardial perfusion with PBS containing heparin. Brains including optic nerves, leptomeninges and choroid plexus were dissected, collected in ice‐cold PBS and cut into small pieces. Tissue was digested in 1 ml of Accutase (Merck Millipore) per brain at 37 °C for 30 min and triturated through 100-μm cell strainers, which were rinsed with 10% FCS in PBS. Cells were purified by a linear 40% Percoll (GE Healthcare) centrifugation step at 650 g without brakes for 25 min and the myelin top layer and supernatant were discarded. Mononuclear cells were resuspended in fluorescence-activated cell sorting buffer (1% BSA and 0.1% sodium azide in PBS) and isolated cells were counted for each brain. For scRNA-seq, cells from the brains of 5 adult (12-month-old), 4 aged (24-month-old), and 4 PLPmut (12-month-old) mice were pooled into 3 separate samples. Viable cells were identified by LIVE/DEAD stain (catalog no. L34965; Thermo Fisher Scientific), Fc receptors were blocked for 15 min with rat anti‐CD16/32 (1:100, catalog no. 553141; BD Biosciences) and cells were washed and labeled with the following antibodies for 30 min at 4 °C: rat anti-CD8 APC (1:100, catalog no. 553035; BD Biosciences); rat anti-CD45 PE (1:100, BioLegend 103105). Cells were washed twice, single viable cells were gated and CD45highCD8+ cells were collected using a FACSAria III and corresponding software (FACSDiva, v.6; BD Biosciences). Around 15,000 CD45highCD8+ single cells  were sorted per sample before being encapsulated into droplets with the Chromium Controller (10x Genomics) and processed according to the manufacturer's specifications. Complementary DNA libraries ready for sequencing on Illumina platforms were generated using the Chromium Single Cell 3′ Library & Gel Bead Kit v2 (10x Genomics) according to the detailed protocol provided by the manufacturer. Libraries were quantified by Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and quality was checked using a 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent Technologies).