alias: E-MTAB-3856:ESC_KO2
title: 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
design: 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
library_construction_protocol: 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.  E14Tg2a wt and Obs3 mutant ESCs were routinely grown in GMEM medium plus 12% FBS (Hyclone) and LIF (Millipore).  Proteinase K treatment and phenol/chloroform purification. Libraries were prepared according to Illumina standard protocols with internal barcodes.
identifiers.namespace: Telethon Institute of Genetics and Medicine (Tigem)
identifiers.id: E-MTAB-3856:ESC_KO2
attributes.tag: Experimental Factor: embryonic stem cell
attributes.value: cell type
attributes.tag: Experimental Factor: Otx2 knockout
attributes.value: genotype