ChIP-qPCR.

ChIP-qPCR was performed as described previously, with some modifications (29). Briefly, cells were transfected with RUNX1-IRES2-AcGFP1, RUNX1-D198G-IRES2-AcGFP1, or RUNX3-IRES2-AcGFP1 and infected with HSV-1 for 5 h at an MOI of 10. After infection, 1% formaldehyde was added to media for 5 min, and then the mixture was neutralized with 0.125 M glycine. The cells were lysed in cell lysis buffer (50 mM HEPES [pH 7.4], 1 mM EDTA, 85 mM KCl, 10% glycerol, 0.5% Nonidet P-40, protease inhibitor) and pelleted at 1,200 × g at 4°C for 5 min. The pellets were suspended in nucleus lysis buffer (50 mM Tris-HCl [pH 8.0], 2 mM EDTA, 150 mM NaCl, 5% glycerol, 1% Triton X-100, 1% SDS, protease inhibitor) and sonicated using a Bioruptor for 45 cycles (30 s on, 30 s off). Samples were incubated with 5 μg of anti-RUNX1 antibody, anti-RUNX3 antibody, or normal rabbit IgG and rotated overnight at 4°C. After incubation with Dynabeads-protein G (Thermo Fisher) for 1 h at room temperature, the beads were washed twice with ChIP wash buffer 1 (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA), twice with ChIP wash buffer 2 (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA), once with ChIP wash buffer 3 (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 500 mM LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA), and once in TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Bound DNA was eluted from beads using a ChIP elute kit (TaKaRa) and analyzed by RT-qPCR with primers flanking RUNX CBSs listed in Table 3 and normalized to a 5% input.

RT-qPCR primers