Tcf3 Targets in Mouse Embryonic Stem Cells E14

To determine the binding targets of Tcf3 in mouse ESCs, ChIP combined with DNA microarray was carried out according to Agilent Mammalian ChIP-on-chip protocol (version 3)3. Briefly, immunoprecipitated (IP) DNA was blunted with T4 polymerase and ligated to linkers with T4 DNA ligase. For reference, 200 ng of input DNA was used. Ligated DNA was amplified with two rounds of PCR cycles. Two ug of each amplified sample were labeled with Cy5 for IP DNA, and Cy3 for input DNA using CGH labeling kit (Invitrogen). Four ug of each labeled sample was blocked with mouse Cot-1 DNA (Invitogen) and blocking agent (Agilent), and hybridized to DNA microarrays (G4490A, Agilent) for 40 h at 65oC. The microarrays were washed and stabilized with acetonitrile (Sigma), followed by stabilization and drying solution (Agilent). The arrays were scanned with Agilent DNA microarray scanner (G2565BA), and probe features were extracted using Feature Extraction Software (G2567AA, version 9.1). The data was then analysed with ChIP Analytics 1.3 (G4477AA), according to the Whitehead Neighbourhood Model, P(Xbar)<0.001, with intra-array median normalization 3. Bound targets which was determined by the algorithm was validated by qPCR on a selected number of genes. The false positive rate was determined. Keywords: ChIP-chip ChIP assays with feeder-free E14 mouse ESCs were carried out as described previously{Zhang, 2006 #49}. Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature and formaldehyde was then inactivated by the addition of 125 mM glycine. Chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using antibodies. The antibodies used for immunoprecipitation were Tcf3 (M-20), HA (Y-11), Tle2 (H-321), Tle3 (M-201), CtBP (H-440), GST (Z-5, all from Santa Cruz). For all ChIP experiments, quantitative PCR analyses were performed in real-time using the ABI PRISM 7900 Sequence Detection System and SYBR Green Master Mix as described previously {Zhang, 2006 #49}. Relative occupancy values were calculated by determining the apparent immunoprecipitation efficiency (ratios of the amount of immunoprecipitated DNA to that of the input sample) and normalized to the level observed. ChIP combined with DNA microarray was carried out according to Agilent Mammalian ChIP-on-chip protocol (version 3) {Boyer, 2005 #28}. Briefly, immunoprecipitated (IP) DNA was blunted with T4 polymerase and ligated to linkers with T4 DNA ligase. For reference, 200 ng of input DNA was used. Ligated DNA was amplified with two rounds of PCR cycles. Two ug of each amplified sample were labeled with Cy5 for IP DNA, and Cy3 for input DNA using CGH labeling kit (Invitrogen). Four ug of each labeled sample was blocked with mouse Cot-1 DNA (Invitogen) and blocking agent (Agilent), and hybridized to DNA microarrays (G4490A, Agilent) for 40 h at 65oC. The microarrays were washed and stabilized with acetonitrile (Sigma), followed by stabilization and drying solution (Agilent). The arrays were scanned with Agilent DNA microarray scanner (G2565BA), and probe features were extracted using Feature Extraction Software (G2567AA, version 9.1). The data was then analysed with ChIP Analytics 1.3 (G4477AA), according to the Whitehead Neighbourhood Model, P(Xbar)<0.001, with intra-array median normalization {Boyer, 2005 #28}. Bound targets which was determined by the algorithm was validated by qPCR on a selected number of genes. The false positive rate was determined.