Nuclei extraction, fluorescence-activated nuclei sorting, and DNA isolation

Total nuclei were isolated as previously described [12] with the following changes. Approximately 100–200 mg of frozen tissue per sample was homogenized in 5 mL of lysis buffer (0.32 M sucrose, 10 mM Tris pH 8.0, 5 mM CaCl₂, 3 mM Mg acetate, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100) by douncing 50 times in a 40-mL dounce homogenizer. Lysates were transferred to a 18-mL ultracentrifugation tube, and 9 mL of sucrose solution (1.8 M sucrose, 10 mM Tris pH 8.0, 3 mM Mg acetate, 1 mM DTT) was dispensed to the bottom of the tube. The samples were then centrifuged at 28,600 rpm for 2 h at 4 °C (Beckman Optima XE-90; SW32 Ti rotor). After centrifugation, the supernatant was removed by aspiration and the nuclear pellet was resuspended in 200 μL staining mix (2% normal goat serum, 0.1% BSA, 1:500 anti-NeuN conjugated to AlexaFluor488 (Millipore, cat#: MAB377X) in PBS) and incubated on ice. Unstained nuclei served as the negative control. The fluorescent nuclei were run through a Beckman Coulter MoFlo Cell Sorter with proper gate settings (Additional file 1: Fig. S1). A small portion of the NeuN⁺ and NeuN⁻ nuclei were re-run on the sorter to validate the purity which was greater than 95%. Only immuno-positive (NeuN⁺) nuclei were collected. DNA was extracted directly from the sorted nuclei without centrifugation using the MasterPure DNA Extraction kit (Epicentre, Madison, WI, USA) following the manufacturer’s instructions.