ChIP-qPCR Quantification Protocol

ChIP experiments were performed as described previously^{71 (link)}, with some modifications. Briefly, 150 ml cell cultures were grown to OD₆₀₀ = 0.8–1.0, cross-linked (1% formaldehyde, 16 min, room temperature), and lysed by bead beating. The chromatin fraction was isolated and sheared to 200–500 bp fragments (30 min, 30 s on/off cycles, 90% amplitude) using a Q800R1 sonicator (QSonica). Immunoprecipitations were performed overnight at 4 °C with 100 µl of 25% slurry of prewashed IgG-agarose beads (Sigma) from lysates corresponding to 80–100 OD₆₀₀ of cells. The beads were washed and eluted, and the eluate was reverse cross-linked at 65 °C for 3 h and incubated with proteinase K for 2 h at 55 °C. DNA was cleaned up with ChIP DNA Clean & Concentrator^TM kit (#D5201, Zymo Research). Immunoprecipitated DNA was quantified by qPCR using primaQUANT SYBR Master mix (Steinbrenner Laborsysteme GmbH. #SL-9902B) and a QuantStudio^TM 3/5 Real-Time PCR system (Applied Biosystems/Thermo Fisher). Primers are listed in Supplementary Table 3. To avoid changes due to different rDNA copy number, the relative fold enrichment for each strain was determined over the average of three rDNA positions (RDN1 #9, RDN1 #12, and RDN1 #25), after normalization to input: [rDNA(IP)/rDNA(WCE)]/[mean(rDNA)(IP)/mean(rDNA)(WCE)]. Graphs were generated using Graphpad Prism and Inkscape.