Epiquik histone extraction kit

Total histone protein was prepared from 8×10⁶ cells using an EpiQuik Histone Extraction Kit (OP-0006; EpiGentek) according to the manufacturer's instructions, and histone protein concentration was measured by the Bradford Dye (500-0006, Bio-rad). A modified protocol was applied for histone extraction of human buffy coat samples. Briefly, 200–300 μl buffy coat samples were incubated with 10 ml hypotonic Tris-HCl solution (10 mM Tris, PH 8.0) for two hours at 4 °C. A protease inhibitor cocktail (P2714; Sigma) was used to prevent protein degradation after lysing the cells. At the end of the incubation, centrifugation was performed for 20 min at 10,000 rpm, 4 °C. The pellet was then mixed with lysis buffer from the EpiQuik Histone Extraction Kit (OP-0006; EpiGentek) and the remaining extraction steps were completed following the manufacturer’s protocol.

Histones were extracted from 100 mg of liver tissue using the EpiQuik Histone Extraction Kit (EpiGentek) following the manufacturer’s protocol. Western blotting was performed for the analysis of asymmetric dimethylation of arginine 3 on histone H4 and total H4 probed with anti-H4R3me2a (1:800, Active Motif) and anti-H4 (1:1000, Abcam), respectively. For measuring histone H3 modification in the liver tissue, we used the EpiQuik H3 modification Multiplex Assay (EpiGentek) following the manufacture’s instructions.