GSM6339291
mice were anesthetized with 2-3% inhaled isoflurane and transcardially perfused with ice cold artificial cerebral spinal fluid (aCSF).  The aCSF was composed of the following (in mmol/L): 126 NaCl, 2.5 KCl, 25 NaHCO3, 1.3 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2 and 12 glucose. Coronal slices from each hemisphere (300μm thick) were cut with a Vibratome 1200 (Leica).  Lateral striatum was dissected from each hemi-section and placed in HABG media (Hibernate A Media, BrainBits, Springfield, IL, HA500; 2% B27 Supplement, ThermoFisher, Cat #17504044; 0.26% Glutamax, ThermoFisher, Cat# 35050061). For each sample (ipsilateral and contralateral), striata from 3 animals was pooled in the same sample for biologic variability.  Tissue was washed by centrifugation and  the tissue was resuspended in 3 mL of 3.5% Collagenase/Dispase (Sigma, USA, 11097113001, 100mg/mL stock) in Hibernate A minus Calcium Media (BrainBits, HACA100) with 0.125% Glutamax.  Samples were then dissociated using the GentleMacs Octo Dissociator (Miltenyi Biotech, Githersburg, MD, 130-195-937) on program 37_NDK_01. After dissociation enzymes were quenched with HABG up to 15 mL. Tubes were spun and the tissue was resuspended in 2 mL HABG media, and 50 uL of Deoxyribonuclease I from bovine pancrease (DNASE) was added. Each sample was triturated about 10 times with a flame polished glass pipette.  Dissociated tissue was then passed through a 100 micron cell strainer (Falcon, 352360), followed by a 40micron cell strainer (Falcon, 352340).  Each strainer was washed with 20mL of HABG media.  Samples were then spun  and resuspended in 4mL of 30% Percol (Sigma, P1644) in Calcium and Magnesium free Hank's Balanced Salt Solution (HBSS, ThermoFisher, 14170112).  One milliliter of HBSS free of calcium and magnesium was then carefully layered on top of the Percol, and layers were allowed to separate for 5 minutes on ice.  Samples were then centrifuged for 15 minutes at 700xg with the brake turned off.  The top layer of myelin and cellular debris was discarded, and the remaining layer was washed with 15mL of HABG media.  Following centrifugation at 400xg for five minutes, the cell pellet was used for fluorescent activated cell sorting (FACS). Cells were inculbated with flourescent antibodies for A2B5, O4 and CD45 and stained with Dapi.  FACS was performed and CD45+ Dapi+ cells were excluded.  Cells positive for A2B5 and/or O4 were collected for scRNAseq. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics)