Nuclei isolation

Nuclei were isolated from iPSNs and postmortem human brain tissue using the Nuclei Pure Prep Nuclei Isolation Kit (Sigma-Aldrich) following the manufacturer’s protocol as previously described (8). Briefly, iPSN lysates were prepared by rinsing iPSNs with 1× phosphate-buffered saline (PBS), adding supplied lysis buffer supplemented with dithiothreitol and Triton X-100 directly to each well, and harvesting iPSNs with a cell scraper. Lysates were transferred to a 50-ml conical tube and vortexed. Sucrose gradients were assembled following the manufacturer’s protocol. A 1.85 M sucrose gradient was used to enrich for neuronal nuclei. Samples were centrifuged at 15,600 rpm and 4°C using a Swi32T swinging bucket rotor and Beckman ultracentrifuge (Beckman Coulter) for 45 min. The supernatant was discarded, and the remaining nuclei pellet was resuspended in 1 ml of supplied nuclei storage buffer to wash the nuclei of any remaining sucrose. Resuspended nuclei were centrifuged at 2500 rpm and 4°C for 5 min. The supernatant was once again discarded, and the resulting nuclei pellet was vortexed in 1 ml of supplied nuclei storage buffer to resuspend for downstream imaging analysis. For Western blots, washed nuclei were lysed in radioimmunoprecipitation assay (RIPA) buffer as described below.