Isolation and Analysis of Minichromosomes

Minichromosomes were isolated essentially as described^{46 (link)}. Cells carrying minichromosome plasmids were grown in 1 L of SILAC media^{70 (link)} (Kaiser SC-Arg-Lys-Trp (Formedium), supplemented with 20 mg/L heavy arginine-HCl (Arg10, CK Isotopes) and 30 mg/L lysine-2HCl (Lys6, CK Isotopes) or, for a control, 20 mg/L light arginine-HCl and 30 mg/L light lysine-2HCl) at 30 °C until log phase and harvested. After washing cells in ice-cold water supplemented with 2 mM PMSF, cells were resuspended in buffer H150 (25 mM HEPES-KOH, pH 7.5, 150 mM KCl, 2 mM MgCl₂, 10% glycerol, 0.02% NP40, 1 mM PMSF, cOmplete Protease Inhibitor cocktail without EDTA (11873580001, Merck), PhosSTOP (4906845001, Merck), 5 mM nicotinamide) and disrupted using a FastPreP-24 bead beater (MP Biomedicals). Clarified lysates were prepared by centrifugation at 20,000 × g for 20 min three times. lacO-containing minichromosomes were isolated by immunoprecipitating LacI-3FLAG with anti-FLAG M2 antibody (12 μg, F1804, Merck) crosslinked to Dynabeads protein G (10765583, Fisher Scientific) by dimethyl pimelimidate. Proteins on purified minichromosomes were eluted in Elution buffer (50 mM ammonium bicarbonate, 0.1% RapiGest SF (186001860, Waters), 1 mM IPTG). The eluted proteins were analysed by SYPRO Ruby staining and western blot analysis.