Chromatin Immunoprecipitation (ChIP) and ChIP-seq

Chromatin Immunoprecipitation experiments were performed according to the Agilent Mammalian ChIP-on-chip manual as described (Singh et al., 2015). Briefly, 1 × 10⁸ ES cells were fixed with 1% formaldehyde for 10 min at room temperature. Then the reactions were stopped by 0.125 M Glycine for 5 min with rotating. The fixed chromatin were sonicated to an average of 200–500 bp (for ChIP-Seq) or 500–1,000 bp (for ChIP-qPCR) using the S2 Covaris Sonication System (United States) according to the manual. Then Triton X-100 was added to the sonicated chromatin solutions to a final concentration of 0.1%. After centrifugation, 50 μl of supernatants were saved as input. The remainder of the chromatin solution was incubated with Dynabeads previously coupled with 10 μg ChIP grade antibodies (ADNP, R&D Systems, AF5919; H3K4me3, Abcam, ab1012; H3K27me3, Abcam, ab192985) overnight at 4°C with rotation. Next day, after 7 times washing with the wash buffer, the complexes were reverse cross-linked overnight at 65°C. DNAs were extracted by hydroxybenzene-chloroform:isoamylalcohol and purified by a Phase Lock Gel (Tiangen, China).

For ChIP-PCR, the ChIPed DNA were dissolved in 100 μl distilled water. Quantitative real-time PCR (qRT-PCR) was performed using a Bio-Rad qPCR instrument. The enrichment was calculated relative to the amount of input as described. All experiments were repeated at least for three times. The relative gene expression levels were calculated based on the 2^–ΔΔCt method. Data were shown as means ± S.D. The Student’s t test was used for the statistical analysis. The significance is indicated as follows: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

For ChIP-seq, the ChIPed DNA were dissolved in 15 μl distilled water. Library constructions and deep sequencing were done by the BGI Shenzhen (Wuhan, China). All ChIP-seq experiments were repeated two times.