α flag agarose

ChIP experiments were done in three biological replications using the protocol and reagents as described previously (Mishra et al., 2007 ; Mishra et al., 2011 (link)). Strains for Cdc7 ChIP were grown in YPD at 25°C and synchronized in G1, S, and G2/M stages of the cell cycle as described for the IP experiments. Strains for Cse4 ChIP were grown in YPD at 25°C to early logarithmic phase. In this culture, we added HU (0.2 M) to synchronize cells in the S-phase or nocodazole (20 µg/ml) to synchronize cells in G2/M stages of the cell cycle, and cultures were incubated at permissive (25°C) and nonpermissive (37°C) temperatures for 2 h. Protein–DNA complexes were captured using α-Flag agarose (A2220, Sigma Aldrich) and α-HA agarose (A2095, Sigma Aldrich), washed, and processed as described previously (Mishra et al., 2007 ; Mishra et al., 2011 (link)). ChIP-qPCR was performed using Fast SYBR Green Master Mix in a 7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA) with amplification conditions as follows: 95°C for 20 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. The enrichment shown as percent input was calculated from three biological replicates of ChIP-qPCR experiments using the _ΔΔC_T method (Livak and Schmittgen, 2001 (link)).