A detailed protocol for ChIP-chip is given in Additional data file 2. Briefly, for each immunoprecipitation approximately 1,000 mid-late gastrula stage embryos (75% to 85% epiboly) were enzymatically dechorionated and then fixed in 1.85% formaldehyde in 1X embryo medium for 20 minutes at room temperature. For conventional ChIP, approximately 200 embryos were used. Glycine (0.125 M) was added to quench the formaldehyde and the embryos were washed in ice cold 1X PBS and snap frozen on liquid nitrogen or used immediately. Fixed embryos were homogenized in lysis buffer and incubated for 20 minutes on ice. Nuclei were collected by centrifugation, resuspended in nuclei lysis buffer then incubated for 10 minutes before diluting with immunoprecipitation (IP) buffer and sonicating the chromatin sample on an ice bath. Sonication conditions were optimized to give fragments of approximately 300 to 700 bp. The lysate was incubated overnight at 4°C with 100 μl of protein G magnetic Dynabeads (Invitrogen, Carlsbad, CA, USA) that had been prebound to 6 μg of the appropriate antibody. Beads were washed five times with RIPA buffer and once with 1X Tris buffered saline (TBS) at 4°C. Bound complexes were eluted from the beads at 65°C with vortexing in elution buffer. Cross links were reversed for 6 hours at 65°C and the chromatin purified by treatment with RNase A, followed by proteinase K digestion and phenol:chloroform:isoamyl alcohol extraction. Three separate ChIP-chip experiments were carried out on three separate batches of embryos.
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