Isolation and Characterization of Neuronal Nuclei

NeuN immunolabeling and FANS were carried out as previously described (82 (link)). Pooled ARH microdissections were dissociated by Dounce homogenization. Nuclei were purified by ultracentrifugation on a 1.8 M sucrose column (100,000 rcf). Pelleted nuclei were collected and stained using rabbit anti-NeuN (1:4000; Millipore, ABN78) followed by Alexa Fluor 488–conjugated goat anti-rabbit immunoglobulin G (1:2000; Thermo Fisher Scientific, A-11008) and the nucleic acid dye TO-PRO-3 as a counterstain (1:1000; Thermo Fisher Scientific). Sorting was performed on the Sony SH800 Cell Sorter (Sony Biotechnology). FSC (Forward scatter) and SSC (Side scatter) gates were set to exclude debris and non-nuclear material, and TO-PRO-3 nuclear gating was used to collect single nuclei only (fig. S1A). Sorted nuclei were pelleted and frozen on dry ice and stored at −80°C. DNA was extracted from sorted nuclei using the AllPrep DNA/RNA Micro Kit (QIAGEN) according to the manufacturer’s directions. RNA was collected from flow-through after DNA isolation using TRIzol LS according to the manufacturer’s instructions (Thermo Fisher Scientific). DNA was eluted from columns using two rounds of 50 μl of nuclease-free H₂O (pH 8.0), dried in SpeedVac (Eppendorf), and then resuspended in 12 μl of TE buffer (pH 8.0). DNA and RNA were quantitated by PicoGreen and NanoDrop, respectively.