Nuclei isolation from mouse cortical tissues was performed as described with minor modifications (Hu* & Fabyanic* et al., 2017; PMID: 29220646). Briefly, mouse brains were rapidly resected on ice. Cortices were flash-frozen in liquid nitrogen and stored at -80°C before nuclear isolation. Nuclei were isolated and purified as previously described with some modifications3. Briefly, 14mL sucrose cushion (1.8M sucrose (Sigma-Aldrich, RNase and DNase free, ultra-pure grade), 10mM Tris-HCl pH 8.0, 3mM MgAc2 (Sigma-Aldrich), and protease inhibitor cocktail (Roche)) was added to the bottom of centrifuge tubes (Beckman Coulter). Using a glass homogenizer (Wheaton), a frozen mouse cortical sample was dounce homogenized (19 times with loose pestle followed by 4 times with tight pestle) in 12mL homogenization buffer (0.32M sucrose, 5mM CaCl2 (Sigma-Aldrich), 3mM MgAc2, 10mM Tris-HCl pH 8.0, 0.1% Triton X-100 (Sigma-Aldrich), 0.1mM EDTA (Invitrogen), protease inhibitor cocktail (Roche)). Homogenates (~12ml) were carefully layered onto the sucrose cushion in the centrifuge tubes, and 10mL homogenization buffer was added atop of the homogenates. The tubes were then balanced and centrifuged in a Beckman Coulter L7-65 Ultracentrifuge at 82,705 g at 4°C for 2hr using a Beckman Coulter SW28 swing bucket rotor (Beckman Coulter). The supernatant was carefully removed via aspiration. 1ml chilled DPBS was added to resuspend the nuclear pellet, and nuclei were subsequently transferred to a 1.5-mL tube. Nuclei were pelleted at 500 g for 10 min at 4C, and then resuspended in 0.01% BSA (Sigma-Aldrich) in DPBS. After resuspension, nuclei were filtered through a 40-μm cell strainer (Fisher Scientific), visually inspected for morphology and quality assurance, and counted using a Fuchs-Rosenthal counting chamber before FANS. All steps of library preparation were performed in a horizontal laminar flow hood (AirCleanⓇ600 PCR Workstation) to minimize environmental DNA contamination. Bisulfite conversion of bulk nuclei was carried out using the EZ DNA Methylation-Direct™ Kit (Zymo, D5020). Bisulfite conversion of single nucleus was carried out using the EZ-96 DNA Methylation-Direct™ Kit (Zymo, Deep Well Format, D5023). PCR tubes containing FANS-isolated nuclei (100-10,000 nuclei), or 96-well plates containing FANS-isolated single nuclei, were heated at 50°C for 20min. 130μL (for bulk) or 32.5μL (for single nuclei) CT Conversion Reagent was added to each well, followed by pipetting up and down to mix. Plates were treated with the following program using a thermocycler: 98°C for 8min, 64°