GSM6339281
Briefly, cells were cross-linked by 1% formaldehyde in the medium for 10 min in room temperature, and then quenched by adding 0.125 M Glycine for 5 min. Collected cells were washed with PBS twice, and then re-suspended in 3.5 ml of sonication buffer. Sonication was performed for 10 cycles with 0.5 min pulse on and 1 min rest, and 24 watts in ice-water mixture. Then cell lysate was spun down with 14,000 x rpm for 10 min at 4 °C. 50 ul of supernatant was saved as input for gDNA. 10 ul of anti-Cas9 antibody (Active Motif) was added and incubate overnight at 4 °C. 50 ul protein G dynabeads was added into antibody-cell lysate mixture and incubate overnight at 4 °C. Then beads were washed with sonication buffer, sonication buffer with high salt (500 mM NaCl), LiCl wash buffer, and TE buffer. Bound protein-DNA complex was eluted from beads by incubation in a 65 °C oven for 15 min, and then reverse cross-linked under 65 °C over-night. The bound DNA was purified with Qiagen QIAquick PCR Purification Kit, and then subject to sequencing. NEBNext® Ultra™ II DNA Library Prep Kit was used