Illumina HiSeq 2000 sequencing; Otx2 binding to Nanog promoter protects embryonal stem cells from corruption of naive pluripotency and is required at morula stage for inner-cell mass differentiation of the epiblast
Otx2 binding to Nanog promoter protects embryonal stem cells from corruption of naive pluripotency and is required at morula stage for inner-cell mass differentiation of the epiblast
ChIP-assay were performed from 10e7 cells per experiment. Cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Glycine at a final concentration of 0.125 M was added to quench the reaction. Cells were collected and lysed in SDS buffer and chromatin was then sonicated to an average size of 0,2-0,3 kb. After a 30 minute step of chromatin preclearing with the Protein A Agarose/Salmon Sperm DNA 50% gel slurry (Millipore), rabbit polyclonal antibody against Otx2 was added to the chromatin and incubated overnight at 4°C. The immunoprecipitated complex was subsequently incubated with the Protein A Agarose for 2-4 hours at 4°C. Agarose beads were then washed, chromatin was eluted and reverse-crosslinked overnight at 65°C.  EpiSCs were induced from ESCs on serum-coated dishes in N2B27 medium supplemented with activin A and Fgf2. Proteinase K treatment and phenol/chloroform purification. Libraries were prepared according to Illumina standard protocols with internal barcodes.