Chromatin immunoprecipitation was performed as previously described (Kaur et al., Disease Models & Mechanisms, 2015). D283 tumorspheres were crosslinked for 10 minutes in 1% formaldehyde and quenched by 0.125M glycine for 5 minutes. Using 500 μl of sonification buffer [50 mM HEPES, pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% (w/v) Triton X-100, 0.1% (v/v) sodium deoxycholate, 1% (w/v) SDS and protease inhibitors] cell pellets were resuspended and sonicated into ∼400 bp with a bioruptor. Samples were incubated with protein A-Dynabeads ovalbumin and salmon sperm for 1 hour at 4°C to preclear the chromatin. OTX2 antibody was coupled to protein A-Dynabeads with protein A (rabbit antibody) in immunoprecipitation buffer. The precleared-chromatin and antibody bound beads were incubated overnight at 4°C. Unbound chromatin was washed away while the bound fraction was eluted in 400 μl of elution buffer and reverse crosslinked with 5 M NaCl (0.2 M final) and 4 μl of RNAse A (1 mg/ml) overnight at 55°C. Samples were treated with proteinase K (20 μg/μl), DNA was extracted using phenol:chloroform and suspended in 50 μl elution buffer 27 NextFlex Illumina Chip-seq kit