Nuclei were isolated from 50-70 mg frozen brain tissue mostly as described in Velmeshev et al. (Science 2019). For each donor, brain tissues from the motor cortex and frontal cortex were dissected with a scalpel blade. The tissues were then homogenized on ice in 5mL lysis buffer (0.32 M sucrose, 3 mM Mg(Ac)2, 3 mM CaCl2, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton-X, and 10 mM Tris-HCl pH 8.0 in DEPC-treated water), containing 0.4 U/μl freshly added Recombinant RNase Inhibitor (RRI; Takara, Cat. # 2313A) until no chunks of tissue were visible in the tissue suspension (~20 strokes) using a glass Dounce homogenizer (Thomas Scientific; Cat. # 3431D76; size A). The homogenized tissues were filtered through a 40 μm strainer (Thermofisher; Cat. # 22-363-547) and transferred to a 30 mL thick polycarbonate ultracentrifuge tube (Beckman Coulter; Cat. # 355631). Nine mL of the sucrose buffer (1.8 M Sucrose, 3 mM Mg(Ac)2, 1 mM DTT, and 10 mM Tris-HCl pH 8.0 in DEPC-treated water) were added to the bottom of the tubes with the tissue homogenate, and the tubes were centrifuged at 34,400 RPM (107,164 FCF) for 2.5 hours at 4°C, using a swing bucket rotor (SW28). The supernatant was aspirated, and the nuclear pellet was submerged on ice for 20 min in 250 uL of DEPC-treated water-based PBS, containing 1% BSA and 0.4 U/μL RRI. The nuclear pellet was then resuspended and filtered twice through a 30 μm cell strainer (Miltenyi Pre-separation Filters; Cat. # 130-041-407). Nuclei were counted using a hemocytometer and diluted to ~2,000 nuclei/μL before performing single-nucleus capture on the 10X Genomics Single-Cell 3' system. The 10X capture and library preparation protocols were used without modification. Matched control, C9-ALS and C9-FTD samples were loaded on the same 10X chip to minimize potential batch effects. Single-nucleus libraries from individual samples were pooled and sequenced on the NovaSeq 6000 machine with an average depth of  ~10,870 unique molecular identifiers (UMIs) per nucleus.