GSM6194085: CTRL-Young; Mus musculus; RNA-Seq
Mice were euthanized in a CO2 chamber following institutional guidelines. Following sacrifice, mouse brains were removed from the skull of the sacrificed animals and placed into a dish containing 1X PBS. Using dissecting microscope, the meninges were manually removed from the brain and placed in a 6 well plate containing 1mL of cell dissociation solution containing DNASE I, 1M HEPES,  Liberase TH (1.2mg/mL) in 1X HBSS. A small magnetic stir bar was placed in each well with the dissected meninges and the plate was placed on a stirring plate at 37 degrees for cell dissociation. The samples were removed from the stirring plate and manually mixed with a P1000 pipette once every 5 minutes until a single cell suspension was formed, approximately 20 min per sample. The single cell suspension was neutralized using DMEM + 10% FBS and the suspension was filtered through a 40uM filter and then centrifuged for 5 min at 10,000 x g. Following centrifugation, the cell pellet was resuspended in 0.4% BSA-PBS and assessed for viability prior to library preparation using trypan blue. Single cell libraries were prepared using 10X Genomics Chromium™ Single Cell 3' Library & Gel Bead Kit v3 as per manufacturer's protocol. For the generation of single-cell gel beads in emulsion, cells were loaded on to the Chromium single cell instrument (10x Genomics) with an estimated targeted cell recovery of ~5,000 cells.