GSM4673431: H3panac_TSA; Danio rerio; ChIP-Seq
Cells were harvested and nuclei were prepared by incubation in buffer, containing 10 mM Tris–HCl (pH, 8.0), 100 mM NaCl, 50 mM KCl, 0.32 M sucrose, 0.5 mM CuSO4 and 1 % Triton X-100. All solutions contained protease inhibitor cocktail (Bimake), and for histone panacetylation analysis 5 mM sodium butyrate was included to inhibit histone deacetylase activity. After 15 min of incubation on ice, the nuclei were pelleted and then resuspended in the same buffer without CuSO4. Next, the nuclei were washed twice with digestion buffer (50 mM Tris–HCl [pH 8.0], 3 mM MgCl2, 1 mM CaCl2, 0.3 M sucrose, and 100 mM NaCl) and treated with 60 U/μl micrococcal nuclease (Thermo Fisher Scientific) for 10 min at 28 °C. The reactions were stopped by the addition of EDTA to a final concentration of 10 mM. The solubilized chromatin was extracted with hypotonic buffer (1 mM Tris–HCl [pH 8.0], 1 mM EDTA). After purification and concentration with Amicon Ultra 30K centrifugal filter devices (Millipore), the chromatin was used for further analysis. Immunoprecipitation reactions were performed overnight at 4 °C in 1 ml of solution containing 50 mM NaCl, 20 mM Tris–HCl (pH 8.0), 5 mM EDTA, 20 mM sodium butyrate, protease inhibitor cocktail and 5 μg of rabbit polyclonal antibodies against modified histones. Histone  H3ac (pan-acetyl) antibody (Active Motif, # 39139), histone H4ac (pan-acetyl) antibody (Active Motif, # 39243) and histone H3K27me3 antibody (Active Motif, # 39155) were used. Rabbit IgG (Jackson ImmunoResearch, #011-000-002) was used as a negative ChIP control. Immune complexes were collected using magnetic beads coated with immobilized protein G (Thermo Fisher Scientific). Immobilized complexes were washed two times for 10 min at 4◦C in low salt buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) and high salt buffer (20 mM Tris–HCl, pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100). Samples were incubated with RNase A (Thermo Fisher Scientific) for 30 min at 37°C. The DNA was eluted from the beads by proteinase K digestion overnight at 55◦C and purified using phenol/chloroform extraction. ChIP samples containing 10 ng of precipitated DNA were prepared for next-generation sequencing using a standard protocol including DNA end repair, A-tailing, and Illumina Y-adapter ligation. Biotinylated probes for targeted enrichment of ChIP-seq libraries (C-TALE) were generated from BACs bearing the locus of interest. We produced hybridization baits using BACs CH211-225L23 and CH211-197P7 (CHORI BACPAC) covering the genomic region chr3: 54945766-55214549.
GEO Accession
GSM4673431