GSM1541978: ChIP-seq_input_excitatory_neurons_rep1; Mus musculus; ChIP-Seq
The neocortex was rapidly dissected from 1-2 adult mice on ice, and the INTACT method was used to affinity purify GFP+ nuclei. Nucleosomes for native ChIP-seq were prepared as previously described [Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Briefly, 1-2 million bead-bound nuclei were digested with 0.025 units/uL micrococcal nuclease (Worthington LS004798) in 500uL of 15mM HEPES pH 7, 1mM KCl, 2mM MgCl2, 2mM CaCl2, 340mM sucrose, 0.15mM spermine, 0.5mM spermidine, and 5mM sodium butyrate at 37°C for 15 minutes. The reaction was terminated by the addition of EGTA to 2mM final concentration. Nucleosomes were extracted for 30 minutes on ice with 200uL 15mM HEPES pH7, 200mM NaCl, 25mM KCl, 2mM MgCl2, 1mM EGTA, 340mM sucrose, 0.15mM spermidine, 0.15mM spermine, and 5mM sodium butyrate. A second 30 minute extraction was performed with the same buffer except the salt concentration was raised to 400mM NaCl. The extracts were combined and dialyzed overnight against 15mM HEPES pH7, 25mM KCl, 1mM β-mercaptoethanol, 1mM PMSF, and 5mM sodium butyrate using a 10K cut-off Slide-a-Lyzer dialysis device (Thermo Scientific 88401). Native ChIP and library construction were combined [Garber M. et al. A high-throughput chromatin immunoprecipitation approach reveals principles of dynamic gene regulation in mammals. Mol. Cell 47, 810-822 (2012); Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Nucleosomes prepared from 0.5-1 million nuclei were incubated with 1ug antibody and 25uL Protein G Dynabeads. The following antibodies were used: rabbit anti-H3K27me3 (Millipore 07-449), rabbit anti-H3K27ac (Abcam ab4729), rabbit anti-H3K4me3 (Abcam ab8580), and rabbit anti-H3K4me1 (Abcam ab8895). Amplified nucleosomal cDNA was fragmented, end-repaired, linker adapted, and sequenced on an Illumina HiSeq 2500 for 50 cycles.