ChIP assay

This assay was performed with the protocol recommended by Millipore. In brief, BMDMs were cross-linked with 1% formaldehyde for 10 min at 37 °C. Cells were lysed in 200 μl of lysis buffer and sonicated on ice with a Branson Digital Sonifier to shear DNA into pieces with an average size of 500 bp. After centrifugation, supernatants were pre-cleared with a 50% salmon sperm DNA/protein A agarose slurry for 1 h. Chromatin was incubated with 2 μg of normal IgG or anti-IRF1 and IRF8 antibodies O/N with rotation. This was followed by incubation with salmon sperm DNA/protein A agarose slurry for 1 h, then samples were sequentially washed with low salt buffer, high salt buffer, LiCl buffer, and twice with TE buffer. After elution, complexes were reverse cross-linked with NaCl for 4 h at 65 °C and digested with proteinase K, EDTA and Tris-HCl for 1 h at 45 °C. Purified DNA was subjected to quantitative real-time PCR with primers. Input DNA (1%) was used for normalization.