Isolation and Sorting of Brain Nuclei

Brain nuclei were isolated as previously described^{13 (link),18 (link)}, with initial homogenization performed with either 1% formaldehyde in Dulbecco’s phosphate-buffered saline or 2 mM DSG (disuccinimidyl glutarate) (ProteoChem) in Dulbecco’s phosphate-buffered saline. Nuclei were stained overnight with PU.1-PE (Cell Signaling 81886S, 1:100), OLIG2-AF488 (Abcam 225099, 1:2,500) or SALL1 AF647 (Thermo Fisher, clone NRNSTNX 51-9279-82, 1:100) or NEUN-AF488 (Millipore MAB 377X, 1:500). Nuclei were washed the following day with 4 ml FACS buffer, passed through a 40 µM strainer, and stained with 0.5 μg ml⁻¹ DAPI. Nuclei for each cell type were sorted with a Beckman Coulter MoFlo Astrio EQ cell sorter and pelleted at 1,600g for 5 min at 4 °C in FACS buffer. Nuclei pellets were snap frozen and stored at −80 °C before library preparation.