GSM6185940
10-week-old male mice were sacrificed by compressed CO2 followed by cervical dislocation. Mouse brains were rapidly dissected, frozen on dry ice and stored at -80°C. To collect prefrontal cortex (PFC) samples, mouse brains were sectioned coronally at Bregma 2.5 and 0.5 with a razor blade in dissection media (20 mM Sucrose, 28 mM D-Glucose, 0.42 mM NaHCO3 in HBSS). The dissected brain tissues were stored at -80 °C until use. Human brains were obtained during autopsies performed at the Basque Institute of Legal  Medicine, Bilbao, Spain. The frozen PFC tissue belonged to a 41-year-old female with a PMI of 22 h. To isolate nuclei, the PFC was placed in 5 ml ice-cold nuclei extraction buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 0.1 mM EDTA, 10 mM Tris-HCl, and 0.1% Triton X-100 with freshly added 50 μl of PIC, 5 μl of 100 mM PMSF and 5 μl of 1M DTT). The tissue was thawed and homogenized by slowly douncing on ice with a loose pestle A (Kimble, 7-ml size) for 15 times, and with a pestle B for 25 times. We filtered the homogenate with a 40-μm nylon mesh cell strainer (Fisher Scientific, 22-363-547) and then centrifuged the sample at 1,000 g for 10 min at 4 °C. We then removed the supernatant and resuspended the pellet in 500 μl ice-cold nuclei extraction buffer.     The suspension was mixed with 750 μl of 50% iodixanol solution (50% iodixanol, 25 mM KCl, 5 mM MgCl2, and 20 mM Tris-HCl (pH 7.8)) and centrifuged at 5,000 g for 15 min at 4 °C. After centrifugation, we followed the s3_WGS protocol1 to perform nuclei fixation and nucleosome depletion. Briefly, the nuclei pellet was resuspended in 5 ml of NIB:HEPES buffer (10 mM HEPES-KOH (pH 7.2), 10 mM NaCl, 3mM MgCl2, 0.1 % Igepal, 0.1 % Tween-20) in a 15 ml centrifuge tube (Corning, 430791). We incubated the mixture at room temperature for 10 min on a rotator (speed: 50 rpm, Argos Technologies RotoFlex) after adding 246 μl of 16 % formaldehyde (Thermo Fisher Scientific, 28908) to the suspension. The mixture was centrifuged at 500 g for 5 min at 4 °C to form nuclei pellet and the supernatant was then removed. 1 mL of NIB:Tris (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1 % Igepal, 0.1 % Tween-20) was then added to resuspend and wash the nuclei pellet to quench the fixation. The nuclei were pelleted by centrifuging at 500 g for 5 min at 4 °C and resuspended in 200 μl 1X NEBuffer 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 µg/ml BSA; NEB, B7202) in a 1.5 ml tube. The suspension was centrifuged at 500 g for 5 min at 4 °C to pellet nuclei which were then resuspended in 760 μl of 1X NEBuffer 2.1. We added 40 μl of 1 % SDS to the nuclei suspension and incubated the mixture at 37 °C for 20 min on a Multi-Therm shaker (Benchmark Scientific, H5000-HC) with a shaking speed of 300 rpm. After SDS treatment, the mixture was centrifuged at 500 g for 5 min at 4 °C. The pellet was resuspended in 1 ml of ATAC-RSB buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2 with freshly added 10 μl of 10% Tween-20). The suspension was pipetted up and down several times and then centrifuged at 500 g for 5 min at 4 °C. Finally, the nuclei were resuspended in 100 μl of ATAC-RSB buffer with freshly added 1 μl of 10% Tween-20. We measured the concentration of the nuclei using a hematocytometer and diluted the nuclei concentration to 5 million/ml using the ATAC-RSB buffer. Library preparation follows Drop-BS protocol. We first completed single-cell lysis, genomic DNA fragmentation, DNA barcoding and bisulfite conversion in droplets. Then we used random priming to add the second adapter to DNA. Finally, we utilized indexing PCR to amplify DNA to obtain sequencing library.