Chromatin immunoprecipitation (ChIP)

ChIP was performed as described⁶¹ with the following modifications. Chromatin from intracellular tachyzoites grown in HFF for 24 h was cross-linked at room temperature for 10 min with 1% formaldehyde. The cross-linked DNA was sheared to average size of 500 bp and was immunoprecipitated using respective antibody at 4 °C overnight. Thirty microliters of 50–50 slurry of Protein G agarose bead was added to the IP and tubes were nutated at 4 °C for an additional hour. DNA was further subjected to a treatment with proteinase K for 2 h and then purified using phenol:chloroform. The control IgG antibody was used as a negative control. Immunoprecipitated DNA from inhibitor treated parasites was quantified and normalized by calculating fold enrichment over nontranscribed telomere DNA⁶².