Rnapii n 20 sc 899

The ChIP assays were based on the SimpleChIP® enzymatic chromatin IP kit (magnetic beads) (Cell Signaling Upstate Biotechnology) and were performed according to the manufacturer's protocol. Briefly, 1.5 × 10⁷ shCtrl (control) or DHX9 knockdown (shDHX9) HeLa cells were mock-infected or infected with the MHV-68 virus at MOI 2 for 12 h. The cells were fixed with 1% formaldehyde, cell extracts were prepared, and chromatin was sheared by sonication. The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG. The bound DNA was quantified by qPCR and expressed as the fold enrichment relative to normal IgG. For re-ChIP experiments, protein–DNA complexes after the first antibody immunoprecipitation were eluted by incubation of the protein G beads with equal volume of 10 mM DTT for 30 min at 37°C. The eluted samples were diluted 40-fold with 1 × ChIP buffer and subjected to immunoprecipitation with the second antibody (31 (link)). The following promoter-specific primers were used: IL6 (F: 5′-cctcaccctccaacaaagat-3′; R: 5′-agttcatagctgggctcctg-3′), IFN-β (F: 5′-gggagaagt gaaagtgggaa-3′; R: 5′-caggagagcaatttggagga-3′), and GAPDH (F: 5′-tactagcggttttacgggcg-3′; R: 5′-tcgaacagga ggagcagagagcga-3′).