GSM5065113: ChIP-seq LucKD Veh Rep1 FOXA1; Homo sapiens; ChIP-Seq
MCF-7 cells were seeded at ~3 x 106 cells per 15 cm diameter plate and treated as described above.  The cells were cross-linked with 1% paraformaldehyde in PBS for 10 minutes at 37°C and quenched in 125 mM glycine in PBS for 5 minutes at 4°C.  The cells were then collected and lysed in Farnham lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM DTT, and 1x RCPIC).  A crude nuclear pellet was collected by centrifugation, resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 7.9, 1 mM DTT, and 1x RCPIC), and incubated on ice for 10 minutes.  The chromatin was sheared at 4°C by sonication using a Bioruptor UC200 at the high setting for four 5-minute cycles of 30 seconds on and 60 seconds off to generate chromatin fragments of ~300 bp in length.  The soluble chromatin was diluted 1:10 with dilution buffer (20 mM Tris-HCl, pH 7.9, 0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl, 1 mM DTT and 1x RCPIC) and pre-cleared with protein A agarose beads.  The pre-cleared supernatant was used in immunoprecipitation reactions with antibodies against the factor of interest or with rabbit IgG as a control.  The immunoprecipitated material was washed once with low salt wash buffer (20 mM Tris-HCl, pH 7.9, 2 mM EDTA, 125 mM NaCl, 0.05% SDS, 1% Triton X-100, 1 mM aprotinin, and 1 mM leupetin), once with high-salt wash buffer (20 mM Tris-HCl, pH 7.9, 2 mM EDTA, 500 mM NaCl, 0.05% SDS, 1% Triton X-100, 1 mM aprotinin, and 1 mM leupetin), once with LiCl wash buffer (10 mM Tris-HCl, pH 7.9, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM aprotinin, and 1 mM leupetin), and once with 1x Tris-EDTA (TE).  The immunoprecipitated material was eluted in elution buffer (100 mM NaHCO3, 1% SDS) and was then digested with proteinase K and RNase H to remove protein and RNA, respectively.  The immunoprecipitated genomic DNA was then extracted with phenol:chloroform:isoamyl alcohol and precipitated with ethanol. The immunoprecipitated DNA was purified further using the MinElute PCR Purification Kit from Qiagen. After purification, 50 ng of ChIP'd DNA for each condition was used to generate libraries for sequencing, as previously described  {Robertson, 2007 #41}, with some modifications.  Briefly, the DNA was end-repaired and a single “A”-base overhang was added using the Klenow fragment of E. coli DNA polymerase.  The A-modified DNA was ligated with Ilumina sequencing adaptors using the Illumina TruSeq DNA Sample Prep Kit.  The ligated DNA (250 ± 25 bp) was size-selected by agarose gel electrophoresis and extraction, amplified by PCR, and purified using AmPure beads (Beckman Coulter).  The final libraries were subjected to QC (size, purity, adapter contamination) and sequenced using an Illumina HiSeq 2000 per the manufacturer's instructions.