GSM3103909: Input_HCT116; Homo sapiens; ChIP-Seq
ChIP was performed using the ChIP-IT High Sensitivity (HS) Kit (Active Motif 53040) according to the manufacturer's recommended protocol with the following modifications as briefly described. Cells were cross-linked with 1% formaldehyde in DMEM culture medium for 10 min at room temperature, and quenched in 0.125 M glycine for 5 min. Cells were then washed twice with phosphate-buffered saline (PBS) followed by 3 times with lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 1 mM PMSF), and resuspended in ChIP Buffer (Active Motif 37516) supplemented with 1 mM PMSF and a proteinase inhibitor cocktail (Sigma-Aldrich P2714-1BTL). Chromatin was sheared by sonication (30 s alternated with 30-s cooling for 25 cycles) using a Bioruptor Pico ultrasonicator (Diagenode). Solubilised chromatin was used for immunoprecipitation by incubation overnight at 4 °C with 5 μg of the polyclonal antibody FL-393 against human p53 (Santa Cruz Biotechnology sc-6243). The immunoprecipitation reactions were transferred to Protein G Agarose Columns (Active Motif 53039) and incubated for 2 h at 4 °C. Then, each column was washed 4 times with 900 μl Wash Buffer AM1. The ChIPed samples were eluted using 100 μl Elution Buffer AM4 at 37 °C. Cross-linking was reversed by adding 5 μl Proteinase K (New England Biolabs P8107S) and incubated overnight at 65 °C. Input and ChIPed DNA were purified using the DNA purification columns. ChIP-seq libraries were prepared using MicroPlex Library Preparation Kit v2 (Diagenode C05010014) and the Agencourt AMPure XP system (Beckman Coulter A63880) was used to select 350–550 bp libraries. Sequencing was carried out on the Illumina MiSeq and HiSeq platforms.