Chromatin Immunoprecipitation of Human Immune Cells

iMonocytes and igranulocytes were purified by MACS sort using CD14 and CD16 beads (Miltenyi Biotec). The purified cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature at a concentration of 15x10⁶ cells/ml. The fixed cells were sonicated for 5 minutes (30 sec on; 30 sec off) using the Diagenode Bioruptor and centrifuged at maximum speed for 10 minutes. Chromatin of 2x10⁵ cells was incubated overnight in dilution buffer (167mM NaCl, 16,7 mM Tris (pH 8), 1,2mM EDTA, 1% Triton X-100) with 1 μg H3K4me3 antibody at 4°C. ProtA/G beads were blocked with dilution buffer (+0,15% SDS) and incubated with the chromatin-Ab for one hour at 4°C. Beads were washed with three different wash buffers and the DNA library was prepared using the Nextera DNA sample prep kit (Illumina) described by Schmidl et al. [19 (link)].