Bioruptor plus

After the stimulation, chromatin immunoprecipitation (ChIP) assay was performed using a ChIP kit (Abcam) as described by the manufacturer’s instruction. In brief, chromatin from crosslinked macrophages was sheared by sonication (8 of 30 s-on and 30 s-off pulses at high power output, Bioruptor Plus, USA) and incubated overnight with rabbit anti-Phospho-Histone H3S10 (Abcam) at 4 °C. Precipitated DNAs were analyzed by RT-qPCR with SYBR Green Premix Ex Taq (Takara) and primers for human IL-8 promoter (−121 to +61) 5′-GGGCCATCAGTTGCAAATC-3′ and 5′-TTCCTTCCGGTGGTTTCTTC-3′, human IκBα promoters (−316 to −15) 5′-GACGACCCCAATTCAAATCG-3′ and 5′-TCAGGCTCGGGGAATTTCC-3′, as well as a human β-actin promoter (−980 to −915) 5′-TGCACTGTGCGGCGAAGC-3′ and 5′-TCGAGCCATAAAAGGCAA-3′.

Proliferating and ICM‐treated IMRO90s were cultured to 80% confluence in 15‐cm plates and they were cross‐linked in 15 mM EGS/PBS (ethylene glycol bis(succinimidyl succinate); Thermo) for 20 min at room temperature, followed by fixation for 40 min at 4°C in 1% PFA. Cells were then processed with the ChIP‐IT High Sensitivity kit (Active motif) according to the manufacturer's instructions. Chromatin was sheared to 200–500 bp fragments via sonication using a Bioruptor Plus (25 cycles, 30 s on/30 s off, high input), immunoprecipitation was done using 4 μg of anti‐HMGB2 antibody (Abcam ab67282) to approx. 30 μg of chromatin and the samples were incubated overnight in a rotor at 4°C. DNA was captured on protein A/G agarose beads and purified using the ChIP DNA Clean & Concentrator kit (Zymo) and used for qPCR. Oligos used in qPCR are listed in Table S4.