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. Throughout the protocol buffers were supplemented with EDTA-free protease inhibitor cocktail and 10 mM of sodium butyrate. For ChIP-Seq homogenates were fixed prior to the addition of 50% iodixanol by adding 1% paraformaldehyde (PFA) for 8 minutes end-to-end rotation. PFA was then quenched by adding 125 μL of glycine for 5 min. 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. Alternatively, for H3K27Ac and H3K4me1 ChIP-Seq, granule cell nuclei from C57BL/6J were sorted by selecting a specific FSC/SSC population identified from GC EGFP+ mice. We observed that this FSC/SSC gated population in granule cell EGFP+ animals resulted > 92% of the sorted nuclei being EGFP positive. Thus, the gate selected allows us to enrich the sample from 65%–70% to 92%–96% on granule cells, as described in (Mellen et al., 2012). After sorting, nuclei were processed using LowCell# ChIP kit. 105 Nuclei were sonicated in a Bioruptor water bath sonicator for 15 min at intervals of 30s on and 30 s off to obtain ~350 bp fragments and chromatin frozen in liquid nitrogen and kept at -80 °C. Each immunoprecipitation (IP) was performed in chromatin aliquots of 5x10e5 nuclei. Immunoprecipitation was performed following LowCell# ChIP kit manufacturer’s instructions, mixing 50:50 IgA and IgG coated beads to 3 μg of antibodies. 1:10 of resuspended chromatin per sonicated nuclei was saved as input sample of the procedure. Immunoprecipitation was performed at 4 °C overnight in an end-to-end rotator. After washing beads with IP DNA, both input and IP samples were digested with Proteinase K 100 μg/ml in the presence of SDS 1% for 2 hr at 50 °C. Then, the lysate was treated with RNase A/T1 mix for 1h at 37 °C to and DNA was extracted with phenol: chloroform: isoamyl alcohol (25:24:1), precipitated in 70% ethanol and dissolved in TruSeq DNA Sample kit resuspension buffer.