Luciferase assays

Cells were grown as described for the ribosome profiling and RNA-seq libraries. At the second media change to fresh LB + glucose, anhydrotetracycline (1 nM) was added and the cells were harvested after another 60 min. Of cultured samples, 45 µl were mixed with 5 µl of phosphate buffer (1 M K₂HPO₄ pH 7.8 and 20 mM EDTA) and frozen on dry-ice. Frozen samples were mixed with 150 µl of Luciferase assay lysis buffer (Cell Culture Lysis Reagent (Promega), 1.25 mg/ml lysozyme, and 2.5 mg/ml BSA) and incubated for 10 min at room temperature. Nanoluc and firefly luciferase activity were detected by Nano-Glo Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Chemiluminescence was monitored by G:BOX (Syngene).