POGZ suppresses 2C transcriptional program and retrotransposable elements in ESCs

ChIP experiments were performed according to the Agilent Mammalian ChIP-on-chip manual as described. Briefly, 1x108 ES cells were fixed with 1% formaldehyde for 10 minutes 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 200-500bp (for ChIP-Seq) or 500-1,000bp (for ChIP-qPCR) using the S2 Covaris Sonication System (USA). For ChIP-seq, chromatin was sheared for 15 min with Peak power:135; Duty factor: 5.0; Cycles/Burst 200. Then Triton X-100 was added to the sheared 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 (FLAG, Sigma, F1804; H3K4me3, Abcam, ab1012; H3K27ac, Millipore, MABE647; H3K9me3 ,Abcam, ab176916; H4K20me3, Cell Signaling Technology, 5737s; TRIM28 SETDB1) overnight at 4℃ with rotation. Next day, after 7 times washing with the wash buffer, the complexes were reverse cross-linked overnight at 65℃. DNAs were extracted by hydroxybenzene-chloroform-isoamyl alcohol and purified by a Phase Lock Gel (Tiangen, China). 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 at least two times. The Cleavage Under Targets and Tagmentation (CUT&Tag) experiments were performed using the 3×Flag-POGZ restoring Pogz-/- mESCs by the Yingzi and Frasergen Company (Wuhan, China) . The detailed methods have been recently described.