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°C for 3.5h, and 4°C forever. Zymo-Spin™ columns or Zymo-Spin™ I-96 Binding Plates were preloaded with either 600μl (bulk) or 250μl (single nuclei) M-binding buffer, respectively. Both bulk and single-cell samples were further purified according to their respective kit instructions, except Low EDTA TE Buffer (IDT) was used as the elution buffer. To perform A3A deamination, 3 µl of denaturing buffer (1.5µl 200mM MES buffer (pH 6.0) with 0.1% Tween-20 and 1.5µl DMSO) was first added to the 9μl eluent retained or each sample. The samples were denatured at 95°C for 1min and snap cooled by transfer to a PCR tube rack pre-incubated at −80°C (for bulk samples) or transfer to an isopropanol dry ice bath and then to the −80°C pre-incubated PCR tube rack (for 96-well plates containing single nuclei). Before thawing, 3 µl of enzymatic reaction mix (1.5µl 200 mM buffer (pH 6.0) with 0.1% Tween-20 and 1.5µl 5μM in-house A3A) was added to each reaction (for a final concentration of 500nM/µl A3A in 15 µl). The deamination reactions were incubated at 37°C for 2h, purified with 1.6x homebrew SPRI beads, eluted in 9µL Low EDTA TE buffer (IDT). To perform random priming and add inline barcodes, 1µL of individual 250µM random primer (with 8-plex inline barcodes: P5L_AD002_H, P5L_AD006_H, P5L_AD008_H, P5L_AD010_H, P5L_AD001_H, P5L_AD004_H, P5L_AD007_H, or P5L_AD012_H) was added to each reaction to allow for subsequent multiplexing reactions during downstream library preparation. Reactions or plates were heated at 95°C using a thermocycler for 3 min to denature and were immediately chilled on ice for 2 min. 10µl enzyme mix (2µl Blue Buffer (Enzymatics B0110), 1µl 10mM dNTP (NEB N0447L), 1µl Klenow (3′→ 5′ exo-) (50U/µl, Enzymatics P7010-HC-L), and 6µl water) was added to each well (a 20µl reaction) and reactions were mixed by vortexing. Plates or reactions were treated with the following program using a thermocycler: 4°C for 5min, ramp up to 25°C at 0.1°C/sec, 25°C for 5min, ramp up to 37°C at 0.1°C/sec, 37°C for 60min, 4°C forever. Next, 2μl Exonuclease 1 (20U/μl, Enzymatics X8010L) and 1μl Shrimp Alkaline Phosphatase (rSAP) (1U/μl, NEB M0371L) was added to each reaction followed by vortexing and incubation in a thermocycler at 37°C for 30min followed by 4°C forever. To multiplex subsequent adaptor ligation reactions, 18.4μl (0.8x) homebrew SPRI beads were added to each reaction and 8 sample/bead mixtures were pooled (up to 24 samples if all inline barcodes were used). The sample/bead mixtures were incubated at room temperature for 5min before being placed on a 96-well magnetic separator (DynaMag™-96 Side Magnet, ThermoFisher 12331D). Supernatant was removed from each well, followed by three rounds of washing with 180μl 80% ethanol. Beads were air dried at room temperature and 10μl Low EDTA TE buffer (IDT) was added to each well to fully resuspend the beads. Eluted samples were then transferred to either new PCR strips (for bulk sample) or 96-well plates (for single nuclei). The reactions were denatured in a thermocycler at 95°C for 3min and subsequently chilled on ice for 2min. 10.5μl Adaptase master mix (2μl Buffer G1, 2μl Reagent G2, 1.25μl Reagent G3, 0.5μl Enzyme G4, 0.5μl Enzyme G5, and 4.25μl Low EDTA TE buffer from xGenTM Methyl-Seq Lib Prep kit, IDT, 10009860) was added to each reaction, followed by vortexing. Reactions were incubated in a thermocycler at 37°C for 30min then 4°C forever. Finally, to amplify the pooled library and add dual indexing barcodes, 30μl PCR mix (25μl KAPA HiFi HotStart ReadyMix, Roche/KAPA Biosystems KK2602, 1μl of 30μM P5 indexing primer, and 5μl of 10μM P7 indexing primer; P5 (D501-508) and P7 (D701-712) indexing primers are listed in Supplementary Sequence 3) were added to each well, followed by mixing with vortexing. Reactions were transferred to a thermocycler programmed with the following stages: 95°C for 2min, 98°C for 30sec, 15 cycles of [98°