GSM4669133: H3K27me3_CUT&RUN; Mus musculus; OTHER
DNA were isolated with Yap, IgG and H3K27me3 antibodies, respectively, and were extracted following the guidelines of Peter J Skene and Steven Henikoff's protocol. Briefly, collected cells were immobilized on Concanavalin A-coated magnetic beads (Bangs Laboratories, BP531), permeabilized with digitonin and incubated with Yap1 (Novus, NB110-58358, 1:50), Tri-Methyl-Histone H3 (Lys27) (Cell Signaling Technology, 9733, 1:50) and anti-rabbit IgG (Millipore Sigma, 12370, 1:50) antibodies separately overnight at 4oC. Protein A/G–Micrococcal Nuclease (pA/G-MNase) fusion protein was added into each sample to a final concentration of 700 ng/mL for 10 min at room temperature after washing away unbound antibodies. Next, the samples were washed with digitonin buffer and added 150 µL digitonin buffer, pre-chilled on ice, and added CaCl2 to a final concentration of 2 mM to activate the pAG-MNase enzyme for DNA digesting. After 2 h digestion at 4oC, the reactions were terminated by 100 uL STOP buffer, and the protein-DNA complexes were released by incubating the tubes at 37˚C for 10 min on a ThermoMixer at 500 rpm. Finally, DNA fragments were extracted by phenol-chloroform and ethanol and quantified by Qubit analysis following the manufacturer's instructions (ThermoFisher Scientific, Q32851). pA/G-MNase was a generous gift from Dr. Steven Henikoff. CUT&RUN libraries were prepared using the SMARTer® ThruPLEX® DNA-Seq Kit (Takara Bio Inc, R400674) following the manufacturer's instructions and sequenced with paired-end 50 bp reads on an Illumina NovaSeq 6000 instrument.
GEO Accession
GSM4669133