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. To profile both 5hmC and true 5mC in the same single cell through physical splitting, bisulfite conversion of single nuclei was first carried out using the EZ-96 DNA Methylation-Direct™ Kit (Zymo, Deep Well Format, D5023). Briefly, 96-well plates containing FANS-isolated single nuclei were heated at 50°C for 20min and stored in -80°C for later use. 32.5μL CT Conversion Reagent was added to each well, followed by sealing the plate and shaking to mix. Plates were treated with the following program using a thermocycler: two cycles of [98°C for 8min, 64°C for 1h45min], and 4°C forever. Purification was performed using Zymo-Spin™ I-96 Binding Plates preloaded with 250μL M-binding buffer according to kit instructions with following modifications. For the Joint-snhmC-seq, 20µl of nuclease-free water was used as the elution buffer. The 9µl eluent retained for the snmC-seq2/split (bisulfite-only) workflow will proceed directly to random priming. To the other 9μl eluent retained for snhmC-seq2/split workflow, 7µl nuclease-free water was added to the eluant, for a total reaction volume of 16µl. The 16µl of bisulfite-treated DNA were denatured at 95°C for 1min and snap cooled by transfer to an isopropanol dry ice bath and then to the −80°C pre-incubated PCR tube rack. To snap-cooled samples, 8µl of deamination enzyme mix (2.4µl of 10x reaction buffer (50mM Bis-Tris (pH 6.0) with 0.1% Triton X-100), 0.48µl BSA, 0.48µl of commercial A3A (at a final concentration of ~500 nM, NEB, E7125L) or in-house A3A (~1000 nM), and 4.64µL nuclease-free water) was added into each well. The deamination reactions were incubated for 30 minutes at 37°C, tapped to mix, and spun down before undergoing an additional incubation for 2.5h at 37°C. Post-incubation, these reactions were purified with 2x commercial SPRI beads (Beckman Coulter B23318), and eluted in 9µl of Low EDTA TE buffer (IDT). For the Joint-snhmC-seq, P5L random primers were added to split single nuclei samples as described for snhmC-seq. Briefly, 1µl of matched, individual 250µM random primers was added to each reaction (9µl) to allow for subsequent multiplexing (up to 24-plex) and pairing of snmC-seq2/split to snhmC-seq2/split reactions of the same single cell. Plates were then heated at 95°C using a thermocycler for 3min to denature and were immediately chilled on ice for 2min. Next, 10µl of enzyme mix (2µl o f10x Isothermal Amplification Buffer II, 0.8µl of Bst 3.0 DNA Polymerase (8,000 U/ml), 1.2µl 100 mM MgSO4 (these three reagents all from NEB M0374L), 2.8µl of 10mM dNTP (NEB N0447L), and 3.2µl water) was added to each well (a 20µl reaction) and reactions were mixed by shaking. Reactions were then incubated at 60°C for 1h. Following this, 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 (D501-508) indexing primer, and 5μl of 10μM P7 (D701-712) indexing primer) 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°C for 15sec, 64°C for 30sec, 72°C for 2min] (for pooled single nucleus reactions), 72°C for 5min, and 4°C forever. For bulk samples, we perform qPCR and subtract two cycles from the inflection point for each sample to determine optimal cycle number for amplification. PCR products were cleaned with two rounds of 0.8x SPRI beads, concentration was determined via QbitⓇ dsDNA High Sensitivity Assay Kit (Invitrogen Q32851), and library size was determined via Bioanalyzer (Agilent High Sensitivity DNA Kit, 5067-4626). Reactions were sequenced on Illumina NovaSeq 6000 (300-cycle v1.5 kit) sequencers.