Histone Extraction from AML12 Cells and Liver

AML12 cell pellets (2 × 10⁶ cells) and liver samples were cut into small pieces (60–70 mg) and transferred to an Eppendorf tube. Thereafter, 1 ml of Triton Extraction Buffer (TEB; PBS containing 0.5% Triton X 100 (v/v), 0.02% (w/v) NaN₃) + inhibitors (Trichostatin 10 μM, nicotinamide 10 mM, sodium butyrate 50 mM, with protease, and phosphatase inhibitors P0044, P5725, and P8340) were added per 200 mg of tissue/1 × 10⁷ cells, and samples were homogenized. Then, lysates were incubated on rotation for 10 min at 4 °C, and the mixture was centrifuged at 2000 rpm for 10 min at 4 °C. The supernatant was removed, and the pellet was resuspended in 100 μl of 0.2 N HCl and incubated on rotation overnight at 4 °C. Afterwards, samples were centrifuged at 2000 rpm for 10 min at 4 °C, and the supernatant was transferred to a new tube. Next, HCl was neutralized with NaOH 0.2 N, and samples were stored at −80 °C until used.