DNA Immunoprecipitation and qPCR Analysis

DRIP was performed as described previously with some modifications [54 (link)]. Genomic DNA was extracted from the indicated strains and digested with Hind III (Neb), EcoR I, BsrG I, Xba I, and Ssp I. The digested DNA was purified and immunoprecipitated with S9.6 antibody (Millipore) in DNA binding buffer (10 mM Na₂HPO₄, 140 mM NaCl, and 0.05% Triton X-100) at 4 °C for 16 h. Protein A+G magnetic beads (Millipore) were used for recovering immunoprecipitated DNA and then washed 3 times with DNA binding buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, pH 8.0, and 0.5% SDS) and twice with TE buffer. The DNA was eluted from magnetic beads (Millipore) with elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, pH 8.0, and 0.5% SDS) and then treated with Proteinase K at 50 °C for 3 h. DNA was purified by phenol/chloroform/isoamyl extraction and then used for qPCR.