The cells were cross-linked with 1% formaldehyde (no. F8775; Sigma-Aldrich, St. Louis, MO) at 37°C for 10 to 15 min; then, the reaction was quenched with 0.125 M glycine for 5 min at room temperature, and the chromatin was sonicated for 30 min in 30-s intervals to shear chromatin into 200– to 1000–base pair lengths. ChIP was performed as described previously (24). Briefly, samples were incubated overnight at 4°C with 3 to 5 μg of antibody bound to 60 μl of Protein A/G PLUS-Agarose; 1 to 2% of precleared chromatin was reserved for use as input DNA before incubation with the antibody. The beads were washed, and the chromatin was eluted; then, the reverse cross-linked ChIP DNA was dissolved in 10 mM tris buffer (pH 8.0). For ChIP-qPCR, immunoprecipitated DNA was analyzed by qRT-PCR; then, the amplification product was expressed as percentage of the input and normalized to the control experiment for each condition. EZH2, H3K27me3, H3K4me3, Smad 2/3, and active β-catenin antibodies were used; ChIP-qPCR primers are listed in table S2.6. DNA libraries from EZH2-ChIP and H3k27me3-ChIP, corresponding to the input DNA samples were prepared and sequenced by BasePair BioTechnology Company (Suzhou, China) with an Illumina Genome Analyzer, as directed by the manufacturer’s protocol.

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