ChIP-seq Protocol for ICP4 Protein

Chromatin immunoprecipitation (ChIP) was performed as previously described [59 (link), 60 (link)] with the following modifications. Per sample, 40 μg of crosslinked DNA (as determined after reverse crosslinking, protease treatment, phenol extraction, and ethanol precipitation) was used as input. ICP4-3xflag crosslinked to chromatin was precipitated by incubation of sheared chromatin with anti-flag M1 agarose affinity gel (Sigma-Aldrich, catalog number A2220) for 3 h at 4 °C. Yields in the eluates were about 3 ng DNA for control samples (untransfected cells) and 10 ng for ICP4-ChIP samples, as determined by measurements with the Qubit DNA HS assay (Thermo Fisher, catalog number Q32851). For qPCR, 10 ng of DNA template for input and 50 pg for eluate samples was used, as described in the “RT-qPCR analysis” section above.