Glycerol-gradient ultracentrifugation

TSSC4-FLAG co-precipitated complexes were eluted from the beads by incubation with 2 × 25 µg of triple FLAG peptide (Thermo Fisher Scientific) for 8 h and overnight at 4 °C. Nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Samples were diluted in gradient buffer (20 mM HEPES/KOH pH 8.0, 150 mM KCl, 1.5 mM MgCl₂) supplemented with Protease Inhibitor Cocktail Set III (Millipore), 0.5 mM PMSF, and 0.5 mM DTT and loaded on a linear 10–30% glycerol gradient. Complexes were fractionated by centrifugation at 130,000×g (32,000 rpm) using SW-40 rotor (Beckman Coulter) for 17 h at 4 °C. Individual fractions (500 µl each, 24 fractions in total) were collected and from every fraction, RNA and proteins were isolated using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol.