Each ChIP-seq experiment on bulk striatal tissue was performed using the striata of four animals and dividing chromatin extracts in four fractions to allow immunoprecipitating same extract with H3K27ac, H3K27me3 and RNAPII antibodies, and including Input controls. Striata of Q140 heterozygous mice and control wild-type (WT) mice at 2 and 6 months were used in the experiments. ChIPseq data were replicated through four independent experiments (experiment 1, using WT and Q140 striatum at 2 months; experiment 2, using WT and Q140 striatum at 2 months; experiment 3, using WT and Q140 striatum at 6 months; experiment 4, using WT and Q140 striatum at 6 months). Male tissues were used in experiments 1 and 3, and female tissues in experiments 2 and 4. Male and female data of same genotype and age were analysed together to determine differentially enriched regions common to both sexes. Single H3K9me3 ChIPseq experiment was performed using the striatum of Q140 and WT male mice of 6 months. For H3K27ac and H3K27me3 ChIP-seq experiments performed on sorted striatal nuclei of WT mice, 250.000 striatal nuclei were used. The data were replicated through two independent experiments. ChIP-seq was performed as previously described3 using antibodies to H3K27ac (ab4729,Abcam), H3K27me3 (C15410195, Diagenode), and RNAPII68. Briefly, for bulk tissue ChIP-seq experiments, pooled tissues were cut into small fragments, fixed in 1% formaldehyde and incubated for 15 min at room temperature. Cross-linking was stopped by the addition of glycine to final concentration 0.125 M. Tissue fragments were washed with cold PBS supplemented with protease inhibitors. The tissues were then mechanically homogenized in sonication buffer to obtain a homogeneous solution. Tissue homogenates or nuclear suspension (see ‘Fluorescence activated cell sorting’ in “Methods” section) were sonicated to obtain DNA fragments <500 bp using Covaris Ultrasonicator E220 and centrifuged. The soluble chromatin fraction was pretreated with protein A Agarose/Salmon Sperm DNA (Millipore) for 45 min at 4 °C. Subsequently, samples were incubated overnight at 4 °C with corresponding primary antibodies. Protein A Agarose/Salmon Sperm DNA was then added and the mixture was incubated for 3 h at 4 °C in a shaker. Agarose beads were washed, protein–DNA complexes were eluted from the beads and de-crosslinked overnight with RNAse A at 65 °C. Proteins were eliminated by 2 h incubation at 45 °C with Proteinase K, and DNA recovered using Qiagen MiniElute PCR Purification Kit.

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