title: GSM2344874: 3A-TAP-D11; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIPs for the TAP-tagged EBNA3s were done as follows. A suspension of 15x10^6 fixed cells was made in 1ml swelling buffer (25M HEPES, pH7.8; 1.5mM MgCl2; 10mM KCl, 0.1% NP-40; 1mM DTT; 1mM PMSF; 1µg/ml aprotinin; 1µg/ml pepstatin A) and incubated at 4oC with rotation for 20 mins. They were then centrifuged at 375g for 5 min at 4oC and the supernatant discarded. The remaining nuclei were re-suspended in 1ml of sonication buffer (50mM HEPES pH7.9; 140mM NaCl; 1mM EDTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% SDS; 1mM PMSF; 1µg/ml aprotinin; 1µg/ml pepstatin A) and incubated on ice for 30 minutes.  Lysate was then sonicated for 1 hour using a Covaris M220 Focused-ultrasonicator with a milliTUBE holder (settings: peak power 75, duty factor 26, cycles/burst 200, set point temperature 6oC). Sonicated lysate was centrifuged at 12000g for 10 minutes at 4oC and supernatant was diluted with 3ml of sonication buffer. From the input sample 5% was kept at 4 oC as a control and the rest was incubated overnight with 16µg of α-FLAG 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 1ng 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).
attributes.tag: GEO Accession
attributes.value: GSM2344874