ChIP analysis (based on Espinosa et al. 2003., and Jallow et al. 2004.)

• Intact embryos (n~500) at stage 8 or stage 12 were fixed in 1% formaldehyde for 30-45 minutes at room temperature.
• Formaldehyde is inactivated by the addition of 125 mM glycine for 30 minutes, followed by two washings in 20% MRS.
• Embryos were homogenized in 5 ml of low salt whole cell extract buffer (25mM Tris-HCl pH 7.5, 70mM KCl, 1mM EDTA, 20% glycerol, 5mM DTT and protease inhibitors) and sonicated six times with 15 seconds pulses on ice with 2 minutes breaks.
• The suspension was cleared by centrifugation for 20 minutes at 14000 rpm
• Supernatants were taken, protein concentration was determined and equalized with IP buffer (50mM Tris-HCl, pH 8, 100mM NaCl, 2mM EDTA, 1mM DTT, 1% NP-40 and protease inhibitors), so the final concentration was 1mg/ml.
• Samples were frozen and stored at –80°C.

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• 1mg of protein was precleared with 40µL of 50% slurry protein A agarose (Sigma), previously washed in IP buffer, for 1-2 hours at 4°C. 
• Precleared proteins were incubated with 2µL of antibody (we use antiSox3c or antiTcf3n antibodies), or antibody together with peptide against which they were raised (control), for 2 hours at 4cC
• add 40µl of 50% slurry protein A agarose, previously saturated with 1mg/ml BSA and 0.3 mg/ml Herring Sperm DNA in IP buffer
• incubate overnight 4°C.
• Wash1 - IP buffer plus 0.1% sodium deoxycholate
• Wash2 - IP buffer with 500mM NaCl
• Wash3 - IP buffer with 250mM LiCl
• Wash twice in TE buffer (10mM Tris, pH 8, 1mM EDTA)
• Elute immunocomplexes and reverse crosslinking in 1%SDS in 50mM Tris-HCl pH8, 1mM EDTA and 200mM NaCl
• digest with proteinase K
• phenol extracted and precipitate DNA.

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• Analyze precipitated DNA sequences using quantitative RT-PCR (qPCR)
• Calculate relative occupancy values and normalize to the level observed in control immunoprecipitation.

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Primers qPCR
5’ Xnr5: CATTGTTGTATTGTTTGATGTTGCTT
3’ Xnr5: TCTTCACACTTATACAGCTTTCATCTGA

5’ Siamois: GGTGGTTCTGCTGCCAAGTTAG
3’ Siamois: CTTGCCCAGAATACTGTCCCAT

5’ Slug: TTGTTTCATGTTTCCATCCCAA
3’ Slug: GGTTAGACCACCCCTAGCTTT