CUT&RUN protocol for K562 cells

Human K562 cells were purchased from ATCC (Manassas, VA, Catalog #CCL-243). CUT&RUN was performed using a centrifugation-based protocol. Ten million cells were harvested by centrifugation (600 g, 3 min in a swinging bucket rotor) and washed in ice cold phosphate-buffered saline (PBS). Nuclei were isolated by hypotonic lysis in 1 ml NE1 (20 mM HEPES-KOH pH 7.9; 10 mM KCl; 1 mM MgCl₂; 0.1% Triton X-100; 20% Glycerol) for 5 min on ice followed by centrifugation as above. (We have found that nucleases in some cells cause Mg⁺⁺-dependent degradation of DNA, in which case 0.5 mM spermidine can be substituted for 1 mM MgCl₂.) Nuclei were briefly washed in 1.5 ml Buffer 1 (20 mM HEPES pH 7.5; 150 mM NaCl; 2 mM EDTA; 0.5 mM Spermidine; 0.1% BSA) and then washed in 1.5 ml Buffer 2 (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 0.1% BSA). Nuclei were resuspended in 500 µl Buffer 2 and 10 µl antibody was added and incubated at 4°C for 2 hr. Nuclei were washed 3 x in 1 ml Buffer 2 to remove unbound antibody. Nuclei were resupended in 300 µl Buffer 2 and 5 µl pA-MN added and incubated at 4°C for 1 hr. Nuclei were washed 3 x in 0.5 ml Buffer 2 to remove unbound pA-MN. Tubes were placed in a metal block in ice-water and quickly mixed with 100 mM CaCl₂ to a final concentration of 2 mM. The reaction was quenched by the addition of EDTA and EGTA to a final concentration of 10 mM and 20 mM respectively and 1 ng of mononucleosome-sized DNA fragments from Drosophila DNA added as a spike-in. Cleaved fragments were liberated into the supernatant by incubating the nuclei at 4°C for 1 hr, and nuclei were pelleted by centrifugation as above. DNA fragments were extracted from the supernatant and used for the construction of sequencing libraries. We have also adapted this protocol for use with magnetic beads (Appendix 3).