GSM4104310: SgriRibo_L1; Streptomyces griseus; OTHER
Thiostrepton (Sigma) was added to cultures to a final concentration of 20 μM to stall the translating ribosomes, and the cultures were incubated for 5 min at 30 °C, and subsequently harvested for the construction of ribosome profiling libraries. Lysate for all samples were prepared by resuspended at polysome buffer and grinding in liquid nitrogen The mycelium treated by thiostrepton was collected by centrifugation at 4 °C for 10 min at 3,000 g, and the cell pellet was washed with 2 ml polysome buffer composed of 20 mM Tris-HCl (pH 7.5), 140 mM NaCl, and 5 mM MgCl2 with 20 μM thiostrepton. The washed pellet was resuspended in 1 ml lysis buffer composed of 950 μl of polysome buffer and 50 μl of 20% triton X-100 with 20 μM thiostrepton. The resuspended cells were dripped into a mortar filled with liquid nitrogen and then ground with pestle. The cell debris was removed by centrifugation at 4 °C for 5 min at 3,000 g. The supernatant was further clarified and collected by centrifugation at 4 °C for 10 min at 16,000 g. To digest RNA, MNase was treated to the samples. Then, the samples were loaded onto illustra MicroSpin S-400 HR Columns. The ribosome-protected RNA fragments (RPF) between 26 and 34 bp were separated by electrophoresis for 65 min at 200 V using 15% polyacrylamide TBE-urea gel (Invitrogen), and eluted in 400 μl of RNA gel extraction buffer. Ribosome profiling