title: ChIP-Seq on Homo sapiens: Primary GBM cells
design: For ChIP-seq experiments we immunoprecipitated HIF-1, TCF1 and TCF4 in either control or Wnt3a-treated (30ng/ml) GBM cells under hypoxic/normoxic conditions. ChIP-seq experiments were performed as previously described 59 with limited modifications. In particular, primary GBM cells were cross-linked and washed. Then, cells were lysed in Lysis buffer 1 (50mM HEPES-KOH, pH 7.5; 140mM NaCl; 1mM EDTA; 10% glycerol; 0.5% NP-40; 0.25% Triton X-100; protease inhibitors) and, after centrifugation, resuspended in Lysis buffer 2 (10mM Tris-HCl, pH 8; 200mM NaCl; 1mM EDTA; 0.5mM EGTA; protease inhibitors). Cells were pelleted and resuspended in Sonication buffer (10mM Tris-HCl, pH 8; 100;mM NaCl; 1mM EDTA: 0.5mM EGTA; 0.1% Na-Deoxycholate; 0.05%N-lauroylsarcosine; protease inhibitors) and sonicated in a Bioruptor sonicator (Diagenode, Denville, NJ). Cell lysates were added to protein G beads (Invitrogen, Carlsbad, CA), previously resuspended in 250l of PBS, 0.5% BSA and 5g of specific antibody, and incubated overnight at 4C.  A small quantity of cell lysate prior to beads addition was kept as Input. Cross-linking was reversed and DNA extracted by a standard Phenol/Chloroform protocol. For library preparation, all samples were prepared by using the Illumina/Solexa Genomic DNA kit (Illumina, San Diego, CA) according to manufacturer's instructions.