GSM8515165: brain, Cd8-cre+, rep2; Mus musculus; RNA-Seq
Mice were anesthetized and sacrificed by cardiac perfusion with ice-cold dPBS (Gibco). Brains were aseptically dissected, minced and enzymatically digested in HBSS containing 0.05% collagenase D (Sigma), 0.1 μg/mL TLCK trypsin inhibitor (Sigma), 10 μg/mL DNase I (Sigma), and 10 mM HEPES (Gibco) at 37°C for 1h with shaking. Tissues were then pushed through a 70 μm strainer and centrifuged at 500× g for 10 min. Brain cell pellets were resuspended in 37% isotonic Percoll (Cytiva, cat. no. 17089101) and centrifuged at 1200× g for 30 min to remove myelin debris. Cells were washed in PBS. Dead cells were removed using the Dead Cell Removal Kit (Miltenyi Biotec, cat. no. 130-090-101). Live cells were diluted to 1000 cells/µL with ice-cold 0.04% BSA in PBS. Samples were sent to Genome Access Technology Center at the McDonnell Genome Institute for library preparation using 10x Genomics Chromium Single Cell 5' Reagent Kits and sequencing.