GSM3334105
GSM3334105: Ribo_T2; Streptomyces avermitilis; OTHER
The cell pellet was washed with polysome buffer (20 mM Tris-HCl pH 7.4, 140 mM NaCl, 5mM MgCl2, and 33.5 ug/mL thiostrepton) and resuspended with lysis buffer (475 uL Polysome buffer, 25 uL Triton X-100, and 6 uL DNase I (New England Biolabs)). The cell suspension was frozen with liquid nitrogen and lysed by grinding using mortar and pestle. The cell lysate was centrifuged at 4 oC for 10 minutes at 16,000 g and soluble supernatant was recovered. Ribosome unprotected RNA was digested by treating RNase I (Invitrogen) by incubating at 37 oC for 45 minutes. After RNase I digestion, RNase activity was inactivated by treating SUPERase-In (Invitrogen) and monosome was recovered using Sephacryl S-400 column (GE Healthcare Life Science). Ribosome protected RNA was recovered using phenol:chloroform:isoamyl alcohol = 25:24:1 solution and rRNA was removed with Ribo-Zero rRNA Removal Kit Bacteria (Epicentre) according to the manufacturer's instructions. After rRNA depletion, RNA was resolved on a 15% TBE-urea gel and 26-34 nt RNA fragments were size-selected. The size-selected RNA was eluted in 300 mM sodium acetate pH 5.2, 1 mM EDTA and 0.25% SDS. The eluted RNA was further purified with ethanol precipitation. Library was constructed with NEB Next small RNA library prep set (New England Biolabs) according to the manufacturer's instructions. The constructed libraries were amplified and indexed using Phusion High-Fidelity DNA Polymerase (Thermo) for Illumina sequencing. The amplification step was monitored on a CFX96 Real-Time PCR Detection System (Bio-Rad) to be stopped before the PCR reaction was fully saturated. The amplified libraries were further size-selected on 2% agarose gel with MinElute Gel Extraction Kit (Qiagen).
GEO
GSM3334105
GEO Accession
GSM3334105