Illumina HiSeq 2500 sequencing; Foxh1/nodal defines context-specific direct maternal Wnt/Beta-catenin target gene regulation in early development
Foxh1/nodal defines context-specific direct maternal Wnt/Beta-catenin target gene regulation in early development
Embryos were cultured in 1/9X MMR at 25°C until the indicated stage and fixed in 1% formaldehyde at room temperature for 45 minutes with gentle rocking. Crosslinking reactions were neutralized by the removal of the formaldehyde solution and incubation with 1ml 0.125M glycine solution for 10 minutes on ice. Embryos were then washed with cold RIPA buffer (50 mM Tris-HCl pH7.4, 150mM NaCl, 1mM EDTA, 0.25% sodium deoxycholate, 1% NP40, 0.1% SDS, 0.5 mM DTT, and Roche cOmplete protease inhibitor cocktail), flash frozen, and stored at -80°C. The fixed embryos were homogenized in RIPA buffer and incubated on ice for 10 minutes. Samples were then microfuged at 14,000 rpm for 15 minutes at 4°C. Pellets were resuspended in RIPA buffer and sonicated on ice using a Branson Digital Sonifier 450 resulting in an average fragment size between 200-500bp. The samples were microfuged at 14,000 rpm for 20 minutes at 4°C to remove insoluble cellular debris. The chromatin was then “pre-cleared” by incubating with Protein A-coated Dynabeads (Invitrogen) for 2 hour at 4°C with rotation.  Antibodies were pre-bound to blocked Protein A Dynabeads by incubating at 4°C for 30 min. A sample of sheared chromatin was frozen for use as an input control. Pre-cleared chromatin was added to antibody-bound Dynabeads, and incubated overnight at 4°C on an end-over-end rotator. The next day, the beads were washed for 20 minutes each with ice-cold ChIP wash solution I (50mM HEPES-KOH pH7.5, 2mM EDTA, 150mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 1mM DTT, and 0.4mM PMSF), ChIP wash solution II (50mM HEPES-KOH pH7.5, 2mM EDTA, 500mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 1mM DTT, and 0.4mM PMSF), ChIP wash solution III (0.25 M LiCl, 1 mM EDTA, 10 mM Tris-HCl pH 8.0, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM DTT, and 0.4 mM PMSF), and TE (10mM Tris, 1mM EDTA, 1 mM DTT, and 0.4 mM PMSF). The DNA was then eluted with TE buffer containing 1% SDS, and reverse-crosslinked at 65°C overnight. The sonicated input control was diluted 3-fold with elution buffer, and also incubated at 65°C. All samples were treated with RNAse A, Proteinase K, phenol/chloroform extracted, and ethanol precipitated overnight. DNA pellets were resuspended in Qiagen EB Solution. ChIP sequencing libraries were prepared using the Nextflex ChIP-seq kit (Bioo Scientific).
Experimental Factor: developmental stage
early blastula stage
Experimental Factor: immunoprecipitate
input DNA
Experimental Factor: protocol
ChIP Seq
Experimental Factor: treatment
NA