Chromatin Binding Assay Protocol

Chromatin binding assay was performed as previously described with modifications [8] (link). Briefly, cells were cultured to an OD₆₀₀ of 0.4, arrested in pre-anaphase (nocodazole), pelleted and washed with 1.2 M Sorbitol. Cells were resuspended in CB1 buffer (50 mM Sodium citrate, 1.2 M Sorbitol, 40 mM EDTA, pH 7.4). Cells were spheroblasted, and resuspended in 1.2 M Sorbitol and frozen in liquid nitrogen. Cells were thawed on ice and supplemented with Lysis buffer (500 mM Lithium Acetate, 20 mM MgSO₄, 200 mM HEPES, pH 7.9), protease inhibitor cocktail (Sigma), and TritonX-100. Lysate was centrifuged at 12,000×g for 15 minutes and supernatant containing soluble fraction and pellet containing chromatin bound fraction were collected and supplemented with 4X Laemelli (Amresco). Whole cell extracts, supernatant, and pellet were resolved by SDS-PAGE and analyzed using c-Myc (9E10) (Santa Cruz), H2B (Santa Cruz), and PGK (Invitrogen).