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uliCUT&RUN was conducted following the published protocols with a few modifications. Briefly, all isolated fresh blastomeres or cells were washed with CUT&RUN wash buffer to avoid possible contamination. The samples were then bound to Concanavalin A-coated magnetic beads, which had been activated and resuspended in CUT&RUN binding buffer. After cell immobilization, bead-bound samples were successively incubated with appropriate amount of primary antibodies against protein-of-interest. After unbound antibodies were washed away, protein A-MNase was added and incubated. After washing, CaCl2 was added to a final concentration of 2 mM to activate pA-MN, and the digestion reaction was carried out. The protein-DNA complex fragments were then released by incubation. After transferring the supernatant to a new tube, SDS and Proteinase K were added and incubated. DNA was then precipitated by phenol/chloroform/isoamylalcohol followed by ethanol precipitation with glycogen and then dissolved in nuclease-free water. Sequencing libraries were prepared using KAPA Hyper Prep Kit following the manufacturer's instructions with slight modifications. Briefly, end repair was conducted followed by end-repair/A-tailing. After adaptor ligation, the DNA fragments were purified by AMPure beads followed by 18 cycles of PCR amplification with KAPA HiFi HotStart Ready Mix. The final libraries were cleaned up with AMPure beads, and all CUT&RUN libraries were sequenced on a NovaSeq6000 platform at Novogene Co., Ltd. with paired-ended 150-bp reads. |
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GENOMIC |
ChIP |
PAIRED
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