GSM3334088
GSM3334088: TSS_TAP_treated1; Streptomyces avermitilis; OTHER
Harvested cells were washed with polysome buffer (20 mM Tris-HCl pH 7.5, 140 mM NaCl, 5 mM MgCl2), and then resuspended with lysis buffer (0.3 M sodium acetate pH 5.2, 10 mM EDTA, 1% Triton X-100). The cell suspension was then 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 the supernatant was stored at -80 oC until used for RNA extraction. RNA was extracted by mixing with equal volume of phenol:chloroform:isoamyl alcohol = 25:24:1 solution. The mixture was then centrifuged and upper aqueous phase was recovered. The RNA was purified with ethanol precipitation. The purified RNA was treated with DNase I (New England Biolabs) to remove any genomic DNA contamination. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Kit Bacteria (Epicentre) according to the manufacturer's instructions. About 700 ng of rRNA-depleted RNA was incubated in 1x RNA 5' polyphosphatase (TAP) (Epicentre) reaction buffer and 1 U of SUPERase-In (Invitrogen) at 37 oC for 1 hour with 1 U of TAP. After ethanol precipitation, 5 pmol of 5' RNA adaptor (5'-ACACUCUUUCCCUACACGACGCUCUUCCGAUCU-3') was ligated to the purified RNA with T4 RNA ligase (Thermo) in 1x RNA ligase buffer and 0.1 mg/mL BSA by incubating at 37 oC for 90 minutes. The adaptor-ligated RNA was then purified using Agencourt AMPure XP beads (Beckman Coulter) according to the manufacturer's instructions. The purified product was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) and purified using Agencourt AMPure XP beads. The purified cDNA was amplified and indexed using Phusion High-Fidelity DNA Polymerase (Thermo) for the 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. Finally, the amplified library was purified using Agencourt AMPure XP beads, and the concentration of the library was measured with Qubit 2.0 fluorometer (Invitrogen). The size distribution of the library was checked with gel electrophoresis on 2% agarose gel.
GEO
GSM3334088
GEO Accession
GSM3334088