Isolation of nuclei from frozen brain tissue.

Tissue sections were snap frozen and stored at −80°C and the nuclei were isolated as previously described^90,91. Briefly, 20 mg frozen tissues were thawed in 1 mL cold homogenization buffer (260 mM sucrose, 30 mM KCl, 10 mM NaCl, 20 mM Tricine-KOH pH 7.8, 1 mM DTT, 0.5 mM Spermidine, 0.2 mM Spermine, 0.3% NP40, complete Protease inhibitor (Roche), and Ribolock) and homogenized in a pre-chilled Dounce. Cell lysates were passed through a 70 μm Flowmi cell strainer before separation using a discontinuous iodixanol gradient and centrifugation at 1480 g at 4°C for 20 min in a swinging bucket centrifuge with the brake off. The nuclei band located at the interface between 30% and 40% iodixanol was collected and washed in RSB-T wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20)