title: GSM1698373: Ribo_S; Streptomyces coelicolor; OTHER
library_construction_protocol: The mycelium was washed in 500 μl polysome buffer composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 20 μM thiostrepton. The cells were then resuspended in the lysis buffer with 20 μM thiostrepton and dripped into liquid nitrogen and then ground with pestle and mortar. The powdered cells were thawed and centrifuged at 4 °C for 5 min at 3,000 g to remove cell debris. The supernatant was recovered and centrifuged at 16,000 g for 10 min to clarify supernatant. To digest RNA, 750 U of RNase I (Ambion) was added to the cell lysate containing 50 μg total RNA in 300 μl of 10 mM Tris-HCl (pH7.6). The samples were incubated at 37 °C for 45 min with gentle rotation, followed by addition of 200 U of SUPERase-InTM (Invitrogen). The digested samples were carefully pipetted onto 900 μl of 1 M sucrose cushion (34% (w/v) sucrose) in a polycarbonate ultracentrifuge tube (13 mm × 51 mm, Beckman). Ribosomes were pelleted by centrifugation with TLA100.2 rotor (Beckman) at 215,000 g for 4 hrs at 4 °C, and subsequently resuspended in 700 μl of RLT buffer (Qiagen). RNA was purified by phenol-chloroform extraction and ethanol precipitation. rRNA was removed by using Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria (Epicentre). The ribosome-protected RNA fragments were separated by electrophoresis using 15% polyacrylamide TBE-urea gel (Invitrogen). A fraction of the fragment between 26 bp and 32 bp was size-selected. Size-selected RNA was then dephosphorylated with T4 PNK (NEB). Universal miRNA Cloning Linker (NEB) was ligated to the RNA. Linker-ligated RNA was reverse transcribed with modified reverse transcription primer. cDNA was then purified by ethanol precipitation and separated from the unextended primer by polyacrylamide gel electrophoresis. Purified cDNA was circularized by using CircLigaseTM ssDNA Ligase (Epicentre). The sequencing library was enriched from the circulated DNA by PCR. PCR amplification was performed with varying numbers of cycles and the optimal cycle was determined from the result. The amplified library was separated from the unextended primers by electrophoresis using 8% polyacrylamide gel.
identifiers.namespace: GEO
identifiers.id: GSM1698373