Ti 41 swing rotor

HCT116 cells were infected with doxycycline-inducible NOP2/NSUN1 shRNA-expressing lentivirus. After puromycin selection, cells were treated with 200 ng/ml doxycycline (Dox) for 4 days before harvesting to induce shRNA expression. Non-Dox-treated cells were used as control. Polysome profile was done as described previously by Simsek et al. (48 (link)). In brief, cells were treated with 100 μg/ml cycloheximide for 5 min and lysed in polysome buffer (25 mM Tris–HCl pH 7.5, 150 mM NaCl, 15 mM MgCl₂, 8% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM DTT, 100 ug/ml cycloheximide, 100 U/ml murine RNase inhibitor (NEB), 25 U/ml TurboDNase, 1× protease inhibitor cocktail). The lysate was cleared by a serial of centrifugation, loaded on 10–50% sucrose gradient, and ultra-centrifuged at 40 000 RPM for 2.5 h at 4°C with a Beckman Ti-41 swing rotor. Fractions were collected using a piston gradient fractionator (BioComp).

To prepare core ribosomal particles and nuclear extracts, nuclei from cells were extracted first as described by Pestov et al. (45 ). Nuclear extracts were prepared by sonication of nuclei in sonication buffer (25 mM Tris–HCl pH 7.5, 100 mM KCl, 2 mM EDTA, 1 mM NaF, 0.05% NP-40) and then centrifugation at 15 000 g for 15 min at 4°C. Core preribosomal particles were extracted from nuclei as described in Lapik et al. (49 (link)). Lysates of nuclear extracts and core preribosomal preparations were loaded on 10–30% sucrose gradient, and ultra-centrifuged at 36 000 RPM for 3 h at 4°C with a Beckman Ti-41 swing rotor. Fractions were collected using a piston gradient fractionator (BioComp). Proteins from each fraction were extracted as described by Pestov et al. (45 ) for western blot analysis. To isolate RNA, fractions were digested with 3.2 U/ml protease K, 1% SDS and 5 mM EDTA at 42°C for 1 h, and extracted with phenol–chloroform.