Polysome Profiling of NOP2/NSUN1 Knockdown

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).