The frozen embryos were ground under liquid nitrogen by mortar and pestle. Chromatin was sheared by the Covaris S2 (20% duty factor, power level 8, 200 cycles per burst) for a total of 30 min processing time (60 sec ON, 45 sec OFF, 30 cycles). To perform the ChIP reactions, extract containing approximately 2 mg of protein was incubated in a microfuge tube with 5 ug of anti-SDC-3 or random IgG antibodies overnight at 4°C. A 25 ul bed volume of protein A sepharose beads was added to the ChIP for 2 hr. ChIPs were washed for 5 min at room temperature twice with FA Buffer (150 mM NaCl), once with FA Buffer (1 M NaCl), once with FA Buffer (500 mM NaCl), once with TEL buffer (10 mM Tris-HCl (pH 8.0), 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and twice with TE Buffer. Protein and DNA were eluted twice with 1% SDS, 250 mM NaCl, 1mM EDTA at 65°C for 15 min. Sequencing libraries were prepared as published in Zhong et al. (2010) with minor changes: sequencing adapters were as described in Lefrancois et al. (2009) and adapters were ligated using NEB Quick Ligation Kit (M2200).