2.7. Chromatin Immunoprecipitation (ChIP) Assays

ChIP experiments were conducted as previously described [20] with some minor modifications. LMH cells were fixed in 1% formaldehyde for 10 min and then with 0.125 M glycine. Each ChIP experiment was performed with sheared chromatin samples from LMH cells (5 × 10⁶ cells) using 5 μg of one of the following antibodies: anti-HA (Sigma-Aldrich, St. Louis, MO, USA, mouse, # H9658) or isotype control IgG1 (Cell Signaling, Beverly, MA, USA, mouse, 5415). Protein A/G PLUS–agarose beads were used for pull-down according to the manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoprecipitated DNA was purified using a QIAquick PCR Purification Kit (QIAGEN, Valencia, CA, USA). The ICP4 promoter region was detected by qRT-PCR using promoter DNA-specific primers with a Bio-Rad CFX96 instrument. Each reaction was performed in triplicate. The primers used for the ChIP-qPCR assay are shown in Table 3.

Primers for ChIP-qPCR of IE genes ICP4 promoter of ILTV.