Bioruptor ucd 200 instrument

All ChIP experiments were performed using the Chromatrap spin column ChIP kit (Porvair). Briefly, 2×10^{6 (link)} cells were crosslinked in their culture dishes with 1% formaldehyde (10 min., room temperature), quenched with glycine, washed twice with ice-cold PBS (containing protease inhibitors), and finally scraped into a microfuge tube. Cell pellets were resuspended in 0.4 ml of hypotonic buffer and incubated for 10 min. on ice. Nuclei were spun down, resuspended in 0.3 ml lysis buffer, and sonicated using a Bioruptor UCD-200 instrument (Diagenode) set to pulse on high (30 sec. followed by 30 sec. rest) for a total time of 10 min. The extracts were centrifuged in a microfuge (top speed, 5 min., 4°C) to remove debris, the supernatants were transferred to new tubes, and stored at −80°C. An amount of extract containing 2 μg of DNA was combined with 4 μg of antibody and loaded on a Chromatrap solid phase Protein A matrix. Immunocomplexes were allowed to form overnight at 4˚C with mild agitation, following which the samples were washed and eluted according to the manufacturer’s protocol. Rabbit IgG and 1% input were used as controls. 1 μl of immunoprecipitated DNA was used in each qPCR reaction.