GSM5859154: D6 GLI1 OE H3K27ac ChIP rep2; Mus musculus; ChIP-Seq
2x106 cells were cross-linked in 1% PFA for 5 minutes at room temperature, with rocking. The reaction was then quenched with 125 mM glycine. The cross-linked tissues were incubated in LB1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) with protease and phosphatase inhibitors on ice for 10 minutes. The tissues were then sonicated in LB3 (10 mMTris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroyl sarcosine, 1% Triton X-100) with protease and phosphatase inhibitors for 5 minutes at 4°C. Chromatin extract was then cleared by centrifugation at 14,000g, 4°C for 10 minutes. To pull-down GLI1-FTA, the chromatin extract was incubated with 50ul M-280 Streptavidin-conjugated magnetic beads (Life Technologies, 11205D) for 30 minutes at room temperature with rotation, washed in sequence by LB3, LB3 with 1 M NaCl, LB3 with 0.5 M NaCl, LB3, and TE. For H3K27ac ChIP, chromatin extract was incubated overnight with 1ul anti-H3K27ac antibody (Wako 306-34849). The next day, immunocomplexes were incubated with 50ul protein G-conjugated dynabeads (ThermoFisher, 10004D) for 4 hours with rotation, before washing as above. The captured chromatin, and input samples, were eluted in ChIP Elution Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% SDS, 250 mM NaCl) at 65°C. After RNase and proteinase K treatment and reverse cross-linking, DNA was purified. High-throughput sequencing libraries from ChIP and input DNA were prepared using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, E7645L). During library preparation, adaptor-ligated DNA fragments of 200-650 bp in size were selected before PCR amplification using Ampure XP magnetic beads (Beckman Coulter, A63880).