title: GSM3494386: RNA-seq over-expression from MdoR sample replicate 2; Escherichia coli; RNA-Seq
library_construction_protocol: Total RNA was extracted using the Guanidium thiocyanate phenol method. RNA integrity was assessed with the Prokaryote Total RNA Nano assay on a 2100 Bioanalyzer (Agilent). Genomic DNA was removed by incubating 10 μg of total RNA with 2U Turbo DNase (Ambion) in a 50 μl final volume for 30 minutes at 37°C in the presence of 10 U SuperaseIn RNase Inhibitor (Ambion). RNA was subsequently phenol-chloroform extracted and purified by ethanol-precipitation. For RNA-seq: Ribosomal RNA was removed with the Ribo-Zero rRNA Removal Kit (Gram-Negative Bacteria) (Illumina) according to the manufacturer's instructions. Successful rRNA depletion was verified on the Agilent 2100 Bioanalyzer. The RNA was fragmented for 5 min at 95°C in the presence of Superscript III buffer (Invitrogen) followed by a five minute incubation on ice. Reverse-transcription (RT) was performed with Superscript III (Invitrogen) in 20 μl reactions according to manufacturer's procedures using 250 ng of ribosomal RNA depleted RNA and 2.5 μM random hexamers (PE_solexa_hexamer, oligo 73, Table S7). The RNA and free primers were degraded using 20U of Exonuclease I (NEB) and 50U RNaseIf (NEB) and the cDNA was purified with the DNA Clean & Concentrator 5 kit (Zymo Research). Ligation of the 5' adapter (P5_phospho_adapter, oligo 39) to the cDNA was performed using CircLigase II (EpiCentre) for 6 hours at 60ºC, followed by a 10 minute inactivation at 80ºC. The cDNA was purified with the DNA Clean & Concentrator 5 kit (Zymo Research). Half of the cDNA library was PCR amplified using Pfu polymerase (Promega) using the P5 forward PCR oligonucleotide and barcoded BC reverse oligonucleotides (200 nM; Table S7; 95°C for 2 min, 95°C for 20s, 52°C for 30s and 72°C for 1 min, and a final extension of 72°C for 5 min. 20 cycles of amplification). The PCR products were treated with Exonuclease 1 (NEB) for 1 h at 37ºC and purified by ethanol precipitation. Libraries were resolved on a 2% MetaPhor agarose (Lonza) gel 200-500 bp fragments were gel-extracted using the MinElute kit (Qiagen). All libraries were quantified on a 2100 Bionalyzer using the High-Sensitivity DNA assay. Individual libraries were pooled in equimolar amounts. Paired-end sequencing (75 bp) was performed by Edinburgh Genomics on a Illumina HiSeq 4000 platform.
identifiers.namespace: GEO
identifiers.id: GSM3494386_2