Fluorescence-Activated Nuclear Sorting

Cell-type specific nuclear purification was performed using fluorescent activated nuclear sorting^{56 (link),57 (link)}. Briefly, frozen striatal tissue was homogenized in ice-cold PBS supplemented with 1× Protease Inhibitors Cocktail (PIC, cOmplete EDTA free, Roche) and cross-linked in 1% formaldehyde for 15 min at room temperature. Cross-linking was stopped by the addition of glycine to final concentration 0.125 M and tissue was washed using ice-cold PBS. Cells were then lysed in Cell Lysis Buffer (10 mM Hepes pH 8; 85 mM KCl; 0.5% NP-40) and nuclei were collected after treatment with Nuclear Extraction Buffer (0.5% SDS, 10 mM EDTA pH 8, 50 mM Tris). Purified nuclei were then resuspended in PBTB (PBS 1×, 5% BSA, 0.5% Tween-20) + 1× PIC, 3% Normal Horse Serum (NHS) and stained using antibody to NeuN (1:1000, Merck Millipore). After washing, nuclei were labelled with Alexa Fluor 488 donkey anti-mouse IgG antibody(1:1500) and washed with ice-cold PBS. Immunostained nuclei were sorted using BD Aria Fusion flow cytometer, recovered in ice-cold 1× PBS, pelleted and stored at −80 °C for posterior ChIP-seq experiments.