Genome-wide binding profiles of Sp8 in differentiation mouse embryonic stem cells
We have determined the genome-wide binding profile of the transcription factor Sp8. We found that Flag-Sp8 prefentially bound to promoters associatd with several growth factor signalling pathways, but most prominently with the Wnt/beta-catenin pathway to selectively promote mesoderm differentiation over neural. These data implicate Sp8 as new regulator of Wnt/beta-catenin target expression to promotes ES cell differentation into the mesoderm lineage. Overall design: Cells were fixed for 15mins in 1% formaldehyde, quenched with glycine. Cells were lysed to extract nuclei which were then sonicated to obtain sheared genomic DNA ~100-300bp in size for sequencing. The libraries were prepared according to standard Illumina's TruSeq Chip protocol. The ChIP DNA is blunt-ended and phosphorylated. A single 'A' nucleotide is added to the 3' ends of the fragments in preparation for ligation to an adapter that has a single-base 'T' overhang. The ligation products are purified and size-selected. The size-selected DNA is purified and PCR-amplified to enrich for fragments that have adapters on both ends. The final purified product is then quantitated by qPCR before cluster generation and sequencing.