Epitope Tagging for Chromatin Immunoprecipitation

Epitope tagging of proteins for ChIP experiments was carried out as previously described (Tran et al. 2017 (link)). Briefly, cells were grown in 50 ml of YEPD (YEP with dextrose) overnight and harvested at an OD₆₆₀ of 0.5. Cells were cross-linked with 1% formaldehyde for 10 min. Chromatin purification was carried out as described (Paeschke et al. 2011 (link)), except that DNA was sheared to an average size of 300 bp using an E220 evolution Focused-ultrasonicator (Covaris, Woburn, MA). Anti-MYC monoclonal antibody (#631206; Clontech) was diluted to 0.02 μg/μl and coupled to 80 μl of Dynabeads protein G (#10004D; Thermo Fisher Scientific). Reverse-cross-linking DNA was performed and purified by QIAquick PCR Purification kit (#28106; QIAGEN, Valencia, CA). Immunoprecipitated chromatin and input DNA were analyzed by quantitative PCR (qPCR) using iQ SYBR Green Supermix (#170–8882; Bio-Rad, Hercules, CA) and a CFX96 real-time system (Bio-Rad). All ChIP experiments were repeated at least three times. Strains and primers are listed in Table S1 and S2. WT cells without a Myc-tagged protein were used as a negative control. Most ChIP-qPCR were quantified by [(ChIP/Input)_{Target site}/(ChIP/Input)_YBL028C]. YBL028C is a control sequence that has very low ScPif1 and Rrm3 binding.