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 harvested at the developmental stage of interest and fixed at room temperature  with  1%  formaldehyde  in  phosphate-buffered  saline  (PBS)  for  45 minutes (1000-cell stage embryos) or for 30 minutes (early gastrulae). Immediately after fixation, the embryos were incubated with 125 mM glycine/PBS for 10 minutes and washed three times with ice-cold PBS for 5 minutes. Batches of 50 embryos were snap-frozen in liquid nitrogen and stored at -80°C for future use. For the following method, all solutions and samples were kept on ice. RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% IGEPAL CA-630, 0.25% Sodium deoxycholate, 0.1% SDS, 0.5 mM DTT) supplemented with Protease Inhibitor Cocktail (Sigma, P8340) was added to frozen embryos. Embryos were thawed on ice for 10-15 minutes, homogenised, and then kept on ice for 10 minutes. After re-homogenisation, the embryo  extracts  were  transferred  to  TPX  microtubes  (Diagenode)  and  sonicated during 2.5 rounds of 10 cycles with 30 seconds ON/30 seconds OFF at high power setting using the Bioruptor Plus Instrument (Diagenode). The sonicated samples were centrifuged at 14,000 rpm for 10 minutes at 4°C, and the supernatant was transferred to a 1.5ml tube for subsequent use for ChIP and input samples. A small aliquot of the supernatant was used for checking chromatin shearing. The input samples were stored at -20°C for later usage. The supernatant for ChIP were incubated for 1 hour at 4°C with  Dynabeads  Protein  G  (Life  technologies)  that  had  been  blocked  with  5% BSA/PBS for 1 hour at 4°C. After snap-spin, the supernatant was transferred to a 1.5ml safe-lock tube and incubated with antibodies (2 ug) overnight at 4°C. Chromatin was precipitated with 5% BSA/PBS-blocked Dynabeads Protein G for 1 hour at 4°C and then the beads were successively washed with ChIP buffer 1 (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), ChIP buffer 2 (20 mM Tris pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), ChIP buffer 3 (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% IGEPAL CA-630, 1% Sodium deoxycholate), ChIP buffer 4 (10 mM Tris pH 8.0, 1 mM EDTA) for 5 minutes each. Chromatin was eluted from the beads with elution buffer  (50  mM  Tris  pH  8.0,  10  mM  EDTA,  1%  SDS)  for  20  minutes  in  a thermoshaker  (65°C,  900  rpm).  At this stage, the frozen input samples were supplemented with elution buffer. ChIP and input samples were incubated with RNase A at 37°C for 30 minutes. The samples were then added with NaCl and incubated for over 16 hours in a thermoshaker (65°C, 900 rpm). The samples were further treated with proteinase K for 2 hours in a thermoshaker (65°C, 900 rpm). The de-crosslinked DNA fragments were purified with phenol:chloroform:isoamylalcohol and precipitated using MinElute Reaction Cleanup Kit (QIAGEN) for sequencing. ChIP sequencing libraries were prepared using the Illumina TruSeq ChIP library preparation kit
Experimental Factor: developmental stage
early blastula stage
Experimental Factor: immunoprecipitate
anti-beta-catenin (Ctnnb1)
Experimental Factor: protocol
ChIP Seq
Experimental Factor: treatment
NA