Polysome Profiling of IAA-Treated C. elegans

L3 larvae grown on regular NGM plates were transferred to NGM plates with and without 1 mM IAA for 24 hours at 20°C. Animals were liquid nitrogen flash frozen in polysome lysis buffer [74 (link)] and ground in liquid nitrogen (with mortar and pestle). The frozen worm powder was thawed on ice and solubilized in polysome lysis buffer that was supplemented with 1 mM DTT, 100 μg/ml cycloheximide, 40 U/100 μl recombinant ribonuclease inhibitor (Invitrogen), 2 U/100 μl DNase (Invitrogen). Lysates were loaded onto 10% to 50% sucrose gradients and spun for 2.5 hours at 40,000 rpm using SW 40 Ti rotor in an ultracentrifugation system (Beckman Coulter). RNA from monosome and polysome peaks was isolated using a density fractionation system (Brandel). The data were used for the analysis in S2C, S2D and S2E Fig.