title: GSM4158372: MCF10A_HRAS_CTCF_1; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were cross-linked for 10 min at 37°C in 1% formaldehyde followed by quenching with 125 mM glycine for 10 min. Fixed cells were lysed with SDS Lysis Buffer (1% SDS, 50mM Tris pH 8.1, 10mM EDTA) supplemented with protease inhibitor and sonicated for 560 seconds (ME220 sonicator, Covaris). Cleared chromatin was diluted 1:10 with dilution Buffer (16.7mM Tris-HCl 8.1; 1.2mM EDTA; 167mM NaCl; 1.1% Triton) and incubated over night with 5 ug α-p53 (DO-1, Santa-Cruz) bound to magnetic beads (Dynabeads Protein G). Chromatin was washed with low salt buffer (20mM Tris-HCl 8.1; 2mM EDTA; 150mM NaCl; 1% triton; 0.1% SDS), high salt buffer (20mM Tris-HCl 8.1; 2mM EDTA; 500mM NaCl; 1% triton; 0.1% SDS), LiCl buffer (10mM Tris-HCl 8.1; 1mM EDTA; 1% NP-40; 250mM LiCl) at 4°C, and twice with TE (10mM Tris-HCl 8.1; 1mM EDTA) at room temp. Complexes were eluted with Elution buffer (10mM Tris-HCl 8.1; 1mM EDTA; 200mM NaCl; 1% SDS). Crosslinks were reversed with 1 mg/mL Proteinase K overnight at 65°C. Purified DNA was used to prepare sequencing libraries using NEBNext UltraII DNA Library Prep Kit (New England Biolabs). Library concentration was measured by DNA High Sensitivity Kit (Invitrogen) on a Qubit fluorometer (Invitrogen). Library quality and fragment sizes were assessed on a Bioanalyzer (Agilent).
attributes.tag: GEO Accession
attributes.value: GSM4158372