Chromatin Immunoprecipitation (ChIP) and Real Time PCR

ChIP and Real Time PCR estimations were performed as described earlier⁷⁵. Cells were grown in YPD at 30 °C and fixed with 1% formaldehyde. Cells were washed thoroughly and lysed mechanically by glass beads in lysis buffer (50 mM K-HEPES pH 7.6, 1% Triton-X 100, 0.1% sodium deoxycholate, 2 mM EDTA, 150 mM NaCl) at 4 °C. Chromatin was sheared to average size of 300 bp by sonication and used for immunoprecipitation with corresponding antibodies (anti-Myc, anti-HA from Millipore) along with Protein A or Protein G agarose beads (GE Healthcare). Beads were washed in wash buffers and eluted at 65 °C in elution buffer (10 mM Tris-Cl pH 8, 2 mM EDTA, 200 mM NaCl, 1% SDS) for 30 min. The ChIP DNA was precipitated using ethanol after deproteinization with phenol, dissolved in TE and quantified in 10 µl reaction mixtures by a real-time PCR method based on SYBR green chemistry using Roche LC480 platform. The subtelomeric region of the right arm of the chromosome VI (TEL VIR) was used as a control region. Occupancies were calculated as the relative enrichment above the control values and normalized against the value for mock immunoprecipitation.

The “Fold enrichment method” was used to calculate the relative enrichments of DNA pol2 over the background using TelVIR as internal control region. In this method, the Ct values obtained from the ChIP and mock samples were first normalized with Ct values obtained from TelVIR region and then by the Ct values of Input⁷⁵. Average and scatter from minimum three independent estimations are plotted. Genes showing non-significant changes with p value > 0.05 are marked with a dot wherever applicable. Rest of the genes have significant p values (<0.05).