title: IRF5 ChIP-seq analysis of CpG-stimulated CD8+ dendritic cells (90 min)
design: CD8-positive dendritic cells were cultured in IMDM-glutamax medium supplemented with 10% FCS, 1% penicillin + streptomycin and 0,1% -mercaptoethanol. Cells were left stimulated with 5 g/ml poly(I:C) HMW or 1 mM class B CpG oligonucleotide 1826 (CpG)  or 10U IFN-beta for 90 min. Stimulated cells were double cross-linked with DSG (40 min) and  formaldehyde (10 min). Nuclei were isolated using lysis buffer containing 150 mM NaCl, 1 nM EDTA pH 8, 20 mM Tris-HCl pH 8, 1% Triton x-100, 1% SDS with EDTA-free protease inhibitor (Roche). Chromtain was sheared with sonication and immunoprecipitated overnight using antibodies against IRF3 (D83B9, Cell Signaling), IRF5 (ab21689, Abcam), IRF9 (AF5629, R&D Systems). Chromatin-antibody complexes were washed and eluted after pull-down by superparamagnetic beads (ThermoFisher Scientific). Eluted complexes were decrosslinked overnight and purified by NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). ChIP-DNA was quantified by Qubit fluorometer. Indexed cDNA libraries were prepared from 1-10 ng ChIP-DNA using TruSeq ChIP Library Preparation Kit (Illumina) according to manufacturers instructions. Libraries were sequenced on NextSeq 500.