title: GSM2058674: HCT116kiTop1_BRD4_+JQ1_ChIPSeq; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP samples were prepared from HCT116, HCT116-siTOP1 and HCT116KI cells following Barski et al. protocol (Barski et al., 2007) with minor changes. Briefly, 5x107 cells were cross-linked with 1% formaldehyde for 10 min. Cross-linking was stopped by the addition of glycine to 125 mM final concentration and cells were washed twice with PBS. After harvesting cells by scraping, the pellet was washed once with PBS plus 0.5% BSA and resuspended in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) supplemented with complete protease inhibitor tablet (Roche) to a final concentration of 1x106 cells/ml. Samples were sonicated 20 times with 20-sec pulses, 30 sec resting, using the Ultrasonic Processor XL (HEAT System) to produce chromatin fragments of 300 bp on average. After clarification by centrifugation, sonicated extracts were adjusted to the conditions of RIPA buffer by adding 1% Triton X100, 0.1% Na-Deoxycholate, 0.1% SDS and 200 mM NaCl.  4 μg of anti-RNAPII (ab817) or anti- TOP1 (ab109374) or anti-BRD4 (A301-985A) were mixed with 40 μl of Dynabeads Protein A (Invitrogen) and incubated at 4°C for 6 hr with rotation. Chromatin from 5×106 cells was added to the Protein A-antibody complexes and incubated overnight at 4°C with rotation. Immunoprecipitates were washed twice with RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 1% Triton X100, 0.1% Na-Deoxycholate, 0.1% SDS, 200 mM NaCl); twice with RIPA buffer plus 300 mM NaCl; twice with LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-Deoxycholate); and twice with TE.  The beads were then resuspended in TE plus 0.25% SDS supplemented with proteinase K (500 μg/ml, Roche) and incubated overnight at 65ºC. After pooling, the DNA was recovered from the eluate by phenol chloroform extraction followed by ethanol precipitation in the presence of 20 μg of glycogen (Roche) and dissolved in TE. The Epicentre DNA END-Repair kit (Epicentre Biotechnologies) was used to generate blunt-ended DNA. DNA was incubated for 45 min at room temperature with a mixture of End repair buffer (33 mM Tris-acetate pH 7, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT), 0.25 mM of each dNTPs, 1 mM ATP, and 1 μl End-Repair Enzyme mix (T4 DNA polymerase + T4 PNK). After purification, the blunt-ended DNA was treated with 15 units of Klenow(exo-) for 30 min at 37°C in the presence of 0.2 mM dATP to generate a protruding 3′ A base used for adaptor ligation. Illumina adapter was ligated to the end of DNA fragments by incubating with 0.1 μl Adaptor oligo mix and 1,000 units of T4 DNA ligase at room temperature for 30 min. After one step of DNA purification using QIAquick PCR Purification Kit the DNA was eluted. A size selection of the adapter ligated DNA was performed through 2% E-Gel (Invitrogen) electrophoresis. The gel slice, around the 200-400 bp region was excised, then DNA was extracted using the MinElute gel extraction kit (QIAGEN) in a final volume of 12 μl elution buffer. The DNA was then amplified for 18 cycles using Illumina primers (Fw: 5'-aca ctc ttt ccc tac acg acg c-3'/ Rv: 5'-caa gca gaa gac ggc ata cga gc-3') according to the following protocol: 98°C for 30 sec; 98°C for 10 sec; 65°C for 30 sec; 72°Cfor 30 sec. The PCR product was obtained by excising 220bps-500bps DNA from a 2.5% agarose gel and purifying it through a Qiagen gel extraction kit (QIAGEN).The purified DNA was used directly for cluster generation and sequencing analysis using the Illumina Genome Analyzer following manufacturer protocols.
attributes.tag: GEO Accession
attributes.value: GSM2058674