title: Yersinia type three secretion system effectors cooperate to suppress the major immune pathways in primary human macrophages
design: Yersinia type three secretion system effectors cooperate to suppress the major immune pathways in primary human macrophages
library_construction_protocol: Macrophages (3–10 x 10⁶ cells per condition, 2–3 biological replicates/macrophage donors per condition) were washed once with warm PBS and incubated for 30 min at 37°C with accutase (eBioscience) to detach the cells. For ChIP, BSA-blocked ChIP grade protein A/G magnetic beads (Thermo Fisher Scientific) were added to the chromatin and antibody mixture and incubated for 2 h at 4°C rotating to bind chromatin-antibody complexes. Samples were incubated for ~3 min with a magnetic stand to ensure attachment of beads to the magnet and mixed by pipetting during the wash steps. Eluted DNA was subjected to ChIP-seq library preparation. Input chromatin DNA was prepared from 1/4 of the chromatin amount used for ChIP. Antibodies used for ChIP were anti-H3S10ph (Invitrogen, 9H12L10, 6 μl (3 μg) per ChIP) and anti-H3K9me3S10ph (Abcam, ab5819, 2 μg per ChIP). ChIP-seq libraries were constructed with 1–10 ng of ChIP DNA or input control as starting material. Libraries were generated using the NEXTflex ChIP-Seq Kit (Bioo Scientific) following the manufacturer's recommendations. Concentrations of all samples were measured with a Qubit Fluorometer (Thermo Fisher Scientific), and fragment length distribution of the final libraries was analyzed with the DNA High Sensitivity Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies). All samples were normalized to 2 nM and pooled equimolar.