Nuclei isolation and sorting were performed as described in (Kriaucionis and Heintz, 2009). Briefly, cerebella were dissected as described above and homogenized in homogenization buffer (0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine [pH 7.8]), 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor cocktail using loose (A) and tight (B) glass-glass dounce. The homogenate was supplemented with 50% iodixanol (OptiPrep), 150 mM KCl, 5 mM MgCl2, 20 mM Tricine (pH 7.8); and laid on a 29% iodixanol cushion. Nuclei were pelleted by centrifugation for 30 min at 10,000 g and at 4 °C in swinging bucket rotor (SW41) in a Beckman Coulter XL-70 ultracentrifuge. The nuclear pellet was resuspended in homogenization buffer and co-stained with DyeCycle Ruby to 20 μM final concentration. A total of 4 cerebella (2 males and 2 females) were pooled before sorting. Nuclei were sorted in a BD FASCAria cell sorter using 635 nm and 488 nm excitation lasers and by gating with two parameters: high GFP signal (compared to wt mice) indicating bacTRAP positive cells and lowest signal for DyeCycle Ruby indicating singlets. After sorting, 3x10e6 nuclei were centrifuged (1200 g 15 min at 4 °C), and resuspended in 500 μL of pre-warmed MNase digestion buffer: 15mM Tris-HCL pH 8, 1 mM CaCl2, 15 mM NaCl, 60 mM KCl, 0.5 mM spermidine. Nuclei were digested with 2.5 U of micrococcal nuclease at 37 °C for 6 minutes. Digestion was stopped on ice by adding 5 mM of EDTA. After spinning (as described in ChIP-Seq) pellet was resuspended in 500 μL of extraction buffer: 10 mM Tris-HCL pH 8, 150 mM of NaCL 0.1% Triton-X 1.5 mM EDTA pH 8 and 0.5 spermidine. Supernatant (S1) was kept at 4 °C after NaCl and Triton-X were adjusted to 150 mM and 0.1% respectively. DNA was mechanically extracted using a 26-ga needle ten times on ice prior to 4 °C end-over-end incubation for 2h. The extract was then centrifuged at 10,000 rpm and 4 °C for 10 min and supernatant was kept as fraction S2. S1 and S2 were combined and 25 μL was reserved as Input. Immunoprecipitation was performed at 4 °C overnight in an end-to-end rotator by combining the extract (S1+S2) with anti MeCP2 antibody (AB1) coated beads (50 μL of protein G and 50 μL of protein A Dynabeads per sample). After incubation, beads were washed in extraction buffer, DNA extracted and library prepared for Illumina sequencing .