GSM2574060: Ribo_L2; Streptomyces tsukubensis; OTHER
For the Ribo-seq experiment, the cells were harvested in the same manner with a translation elongation inhibitor thiostrepton (Sigma) added to the cultures to a final concentration of 20 mM. Thiostrepton treated cell cultures were then subsequently incubated for 5 min at 30 oC, and then harvested. Cell growth was monitored by measuring the OD600 nm. Collected mycelium culture was at 3,000 x g for 10 min at 4 oC. The cell pellets were resuspended in 2 mL Polysome buffer (20 mM Tris-HCl (pH 7.4), 140 mM NaCl, and 5 mM MgCl2). The resuspended cells were centrifuged at 3000g for 10 min. The cell pellets were then resuspended in 1 mL lysis buffer (20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 1% Triton X-100). The cells resuspended in the lysis buffer were dripped into a mortar filled with liquid nitrogen, and grinded by pestle. The grinded cells were thawed, and centrifuged at 3,000g for 10 min at 4 oC for removal of cell debris. The supernatants were further cleaned by centrifugation at 16,000g for 10 min at 4 oC. For Ribo-seq,Concentration of the lysate was measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). To digest RNA, 50 µg of lysate was incubated at 37 oC for 45 min with 750 U of RNaseI (Invitrogen). The digested samples loaded on equilibrated Sephacryl S 400 MicroSpin column (GE Healthcare) with 500 µL polysome buffer three times. Column was centrifugated at 4 oC for 2 min at 400g. Flow through were gathered and further purified by phenol-chloroform extraction and ethanol precipitation. Ribosomal RNA was removed by using Ribo-ZeroTM kit. The ribosome-protected RNA fragments were separated by gel electrophoresis for 60 min at 200 V on 15% polyacrylamide TBE-urea gel (Invitrogen). RNA fragments between 26 bp and 34 bp were size-selected and eluted in 400 µL of RNA gel extraction buffer (300 mM sodium acetate (pH5.5), 1 mM EDTA, and 0.25% (w/v) SDS). Samples were frozen at -80 oC for 30 min, followed by incubated at RT for overnight with gentle mixing. RNA fragments were purified by ethanol precipitation and phosphorylated as follows: samples were denatured at 80 oC for 90 sec, followed by equilibrated to 37 oC and incubated at 37 oC for 1 h with 10X T4 PNK buffer (New England Biolabs), 20 U of SUPERase InTM (Invitrogen), and 10 U of T4 PNK (NEB).