Cell Cycle Synchronization and Fractionation

For cell-cycle synchronization, mESC (J1) cells^{124 (link)} were treated with 1.25 mM Thymidine for 14 h and then 50 ng/mL Nocodazole for 7 h. G1 and S phase cells were collected at 1.5 h and 7 h, respectively, after Nocodazole release. mESCs were treated with DRB (100 μM) and ActD (1 μg/mL) for 3 h to inhibit transcription. For heterochromatin fractionation, sucrose gradient centrifugation of mESC nuclear extracts was performed as previously reported with modifications.^{125 (link),126 (link)} For embryonic microinjection, ASO (5 μM) and AMO (1 mM) was injected into PN3 zygotes on a Leica DMI3000B microscope equipped with a Leica micromanipulator.