title: GSM3384452: Input1; Homo sapiens; ChIP-Seq
library_construction_protocol: 15 x 10^6 cells were fixed for 10 mins in 1% formaldehyde at room temperature and then washed, resuspended in swelling buffer (25 M HEPES, pH 7.8; 1.5 mM MgCl2; 10 mM KCl, 0.1% NP-40; 1 mM DTT; 1 mM PMSF; 1 μg/ml aprotinin; 1 μg/ml pepstatin A) and incubated at 4oC for 20 mins with rotation. The swelling buffer was aspirated after centrifugation. Τhe pelleted nuclei were resuspended in 1 ml sonication buffer (50 mM HEPES pH 7.9; 140 mM NaCl; 1mM EDTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% SDS; 1 mM PMSF; 1 μg/ml aprotinin; 1 μg/ml pepstatin A), incubated on ice for 30 mins and then sonicated for 1 hour with a Covaris M220 Focused-ultrasonicator with milliTUBE holder (peak power 75; duty factor 26; cycles/burst 200; temperature 6oC). The lysate was centrifuged at 10,000g for 10 mins at 4oC and the 1 ml supernatant was diluted with 3.2 ml sonication buffer. 200 μl of the sonicated lysate were taken for input control. Lysates were incubated overnight with 16µg of the relevant antibody and 120µl of ChIP-grade protein G magnetic beads (NEB, #9006) at 4oC in a 15ml Falcon tube on rollers. The following day the beads were washed with standard ChIP wash buffers, 4ml buffer for each wash for 15 minutes at 4oC on rollers. Precipitated chromatin was eluted in 400µl of elution buffer, the eluate was treated with RNase, formaldehyde cross-links were reversed before proteinase K treatment and DNA was cleaned with a Qiagen PCR purification MinElute kit, for ChIP sample and input control. DNA from ChIP was run on a 2% agarose gel (Bio-Rad Low Range Ultra, #161-3107) and DNA between 100-500bp was excised and purified using the Qiagen MinElute gel purification kit, according to manufacturing instructions. At least 5ng of DNA for each sample was then sent to the Harvard Biopolymers facility for library construction (ChIP-Seq Wafergen) and sequencing (Illumina HiSeq 2500, 50bp single-reads).
identifiers.namespace: GEO
identifiers.id: GSM3384452