ChIP-seq analysis of Brachyury (T) in invitro derived NMPs

Brachyury (Tbxt or just T) is a T-box transcription factor and known target of the cnaonical Wnt signalling pathway. Brachyury is an important gene in the regulation of neuromesodermal progenitors, a stem cell-like population of cells that drive axial elongation during mouse embryogenesis. The goal of the project was to investigate the binding pattern of brachyury in in vitro derived NMPs. Overall design: ES cells were differentiated in serum free N2B27 media and treated with bFGF (12ng/ml) from Day0 to Day2 followed by 3uM CHIR and 12ng/ml bFGF from Day2 to day3. Cells were harvested and fixed in 1% formaldehyde solution for 15min, quenched in 0.125M glycine and flash-frozen. Nuclear extracts were incubated with Goat IgG and anti-T/Brachyury antibodies (AB-108-C, AF2085, R&D systems, Inc respectively) for chromatin immunoprecipitation followed by sequencing.