Isolation of Neuronal Nuclei by FANS

Single neuronal nuclei were isolated using fluorescence-activated nuclear sorting (FANS) for NeuN, as described previously^{6 (link),38 (link)}. Briefly, nuclei were prepared from unfixed frozen human brain tissue, previously stored at −80°C, in a dounce homogenizer using a chilled tissue lysis buffer (10mM Tris-HCl, 0.32M sucrose, 3mM Mg(Oac)2, 5mM CaCl2, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, pH 8) on ice. Tissue lysates were carefully layered on top of a sucrose cushion buffer (1.8M sucrose 3mM Mg(Oac)2, 10mM Tris-HCl, 1mM DTT, pH 8) and ultra-centrifuged for 1 hour at 30,000 x g. Nuclear pellets were incubated and resuspended in ice-cold PBS supplemented with 3mM MgCl2, filtered (40 μm pore size), then stained with Alexa Fluor 488-conjugated anti-NeuN antibody (Millipore MAB377X). Large neuronal nuclei were then subjected to FANS, one nucleus per well into 96-well plates.