ChIP-seq Protocol for dCas9 Protein

Cells expressing gDNA and in control (transfected with GAL4 plasmid) groups were all expanded to 150 mm culture dishes, crosslinked with 2% formaldehyde, and quenched with 0.25 M glycine^{16 (link)}. Cells were treated with cell lysis buffer and nuclear lysis buffer to isolate chromatin. Then the separated chromatin was washed with the mixture of 8 M urea, re-suspended in the IP-binding buffer, and sonicated into ~500 bp length of segments. Hydrophilic streptavidin magnetic beads (NEB, Ipswich, MA, USA) were added into the IP-binding buffer and rotated overnight at 4 °C to isolate dCas9 protein. Isolated proteins were subjected to western blot analysis and SDS-PAGE. Proteins in SDS-PAGE gels were visualized after silver staining^{15 (link),16 (link)}.