title: GSM1698360: TSS_TEX; Streptomyces coelicolor; RNA-Seq
library_construction_protocol: RNA samples from various conditions were pooled to make 10 μg of total RNA. Genomic DNA was removed by using DNA-freeTM Kit (Ambion), and rRNA was removed by using Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria (Epicentre). Total mRNA was split into two samples for two different libraries: the library of the primary transcriptome and the library of whole transcriptome. TerminatorTM 5’-Phosphate-Dependent Exonuclease (TEX, Epicentre) was used to treat one of the samples to enrich primary transcripts which have triphosphate at their 5’-ends, resulting in the preparation of the TEX-treated (TEX+) and non-treated (TEX-) samples. The triphosphates at the 5’-ends of mRNA were converted to monophosphate by treating RNA 5’-polyphosphatase (Epicentre). The mRNA was then purified by phenol-chloroform extraction and ethanol precipitation. 5’-RNA adaptor was ligated to the purified mRNA, then cDNA was synthesized using random 3’ overhanging primer (N9). Synthesized cDNA was purified using QIAquick PCR Purification Kit (Qiagen). The cDNA was purified again by ethanol precipitation. Purified cDNA was selected at a size range between 100 bp and 350 bp on a 2% agarose gel by Pippin Prep (Sage Science). Then the purified sequencing library was amplified by PCR with indexed primers for Illumina sequencing platform. After the enriched library was purified by ethanol precipitation, the library of size between 150 bp and 400 bp was extracted. The size-selected library was purified by ethanol precipitation. A second PCR amplification was carried out with a few PCR cycles to produce enough amount of library for Illumina sequencing. The final amplified library was purified by ethanol precipitation and those of size 150 bp to 400 bp were extracted from 2% agarose gel after electrophoresis. The final library was then purified using MinElute Gel Extraction Kit.
identifiers.namespace: GEO
identifiers.id: GSM1698360