GSM2065371: TEX-_E. coli O104:H4; Escherichia coli O104:H4; RNA-Seq
Total RNA was extracted using the Trizol reagent (Thermo Fisher Scientific) and the concentration and purity of the samples were determined using Nano Drop. RNA integrity was monitored using the R6K ScreenTape system on the Agilent 2200 TapeStation. gDNA was removed by Turbo DNase (Thermo Fisher Scientific) in the presence of 1U/L RiboLock RNase Inhibitor (Thermo Fisher Scientific) for 60 min at 37°C. Following organic extraction (1x 25:24:1 v/v phenol/chloroform/ isoamyalcohol, 1x chloroform), RNA was recovered by overnight precipitation with 3 volumes of 30:1 100 % ethanol/3 M sodium acetate (pH 5.2). Next, the sample (TEX+) was depleted of processed RNAs using 1U per 1μg of RNA Terminator 5´-Phosphate-Dependent Exonuclease (TEX, Epicentre) in the presence of 1U/μL RNase Inhibitor for 60 min at 30° C. A control reaction without TEX (TEX-) was run in parallel. Following organic extraction, RNA was recovered by overnight precipitation and resuspended in RNase-free water. cDNA libraries for the Illumina sequencing platform were constructed by vertis Biotechnology AG, Germany, as described previously (Berezikov et al.,2006), without the RNA size-fractionation step prior to cDNA synthesis. Briefly, RNA samples were polyA-tailed using polyA polymerase. Then, the 5'-PPP termini were converted to 5'-P using tobacco acid pyrophosphatase (TAP) to allow for the ligation of the 5’ end RNA adapter. First-strand cDNA was synthesized by an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. In a PCR-based amplification step using a high fidelity DNA polymerase the cDNA concentration was increased to 20-30 ng/µl. A library-specific barcode for multiplex sequencing was part of a 3'-sequencing adapter.
GEO Accession
GSM2065371