Ez prep

Mice were anesthetized with 3% isoflurane and brains were removed and placed into ice-cold oxygenated artificial cerebrospinal fluid (aCSF). SC were dissected from the midbrain and placed into RNAlater (Invitrogen, AM7021) and stored at 4°C overnight. To ensure the quality of the experiment, two replicates were conducted and five mice were used for each replicate. On the day for the experiment, tissue samples were washed with phosphate buffered saline (PBS) (Gibco, REF 10010–023) and cut into pieces <1 mm and were homogenized using a glass Dounce tissue grinder (Sigma, Cat# D8938) in 2 ml of ice-cold EZ PREP (Sigma, Cat# NUC-101). Then the nuclei suspension was transferred into a 15 ml tube and incubated on ice for 5 min with 2 ml of ice-cold EZ PREP added. After incubation, the nuclei were centrifuged at 500× g for 5 min at 4°C. The nuclei were re-suspended with 4 ml ice-cold EZ PREP and incubated on ice for another 5 min. Then the nuclei were centrifuged at 500× g for 5 min at 4°C and washed in 4 ml Nuclei Suspension Buffer (NSB; consisting of 1× PBS, 0.04% BSA, and 0.1% RNase inhibitor [Clontech, Cat# 2313A]). After being re-suspended in 2 ml NSB, the nuclei were filtered with a 35 μm cell strainer (Corning, Cat# 352235). The nuclei density was adjusted to 1,000,000 nuclei/ml and placed on ice for use.