GSM5418864: Wild type-input-M2; Mus musculus; ChIP-Seq
Wild type and AHR knockout colon organoids grown in WENR condition were stimulated with 5nM FICZ for 1 hour in a glass vial at 37oC. Samples were crosslinked with 1% formaldehyde (catalogue number: 28906; Thermofisher) for 8 minutes and quenched with 0.125M glycine at room temperature. Nuclei was extracted by Dounce homogenization and chromatin was sheared by sonication in a lysis buffer (0.25% SDS, 1mM EDTA, 1% Trition-X, 10mM Tris-HCl, pH 8). Debris was spin pelleted at 13,000 rpm for 30min at 4oC. Supernatant was diluted in RIPA buffer (0.2% deoxycholate, 280mM NaCl, 1.1% Triton-X in H2O) and cleaned with protein A/G Dynabeads (10003D & 10001D; Invitrogen) for 1 hour at 4oC. 10% of supernatant was used as input and the remaining sample was immunoprecipitated overnight at 4oC with anti-AhR antibody (BML-SA210; Enzo). Chromatin was then incubated with protein A/G Dynabeads for 3 hours at 4oC. Chromatin bound beads were washed twice in low-salt buffer (0.1% SDS, 1% Triton X-100, 1mM EDTA, , 140 mM NaCl, 0.1% deoxycholate, 20 mM Tris–HCl, pH 8), high-salt buffer (same as low salt except for 500 mM NaCl), LiCl wash buffer (250mM LiCl, 0.5% NP40, 0.5% deoxycholate, 1 mM EDTA, 10 mM Tris–HCl, pH 8) and with Tris-EDTA buffer (10 mM Tris–HCl 1mM EDTA). Protein-chromatin complexes were then eluted in elution buffer (1% SDS, 100 mM NaHCO3) and reverse cross-linked overnight with RNase and proteinase containing buffer (62.5 µg/ml RNaseA, 5 mg/ml proteinase K, 1.25M NaCl, 62.5mM EDTA pH 8, 250mM Tris-HCl pH 6.5). ChIP DNA was purified using the ChIP DNA Clean and Concentrator (D5205, Zymo Research) and quantified using Bioanalyzer followed by sequencing. Libraries were prepare for sequencing using KAPA_Hyper_kit_ChIP