GSM5060699: WT_R1_SE; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
RNAs were extracted according to the FastRNA Pro protocol (Qbiogene). The rRNA-depleted RNA samples (RiboZero) were fragmented with ultrasound (4 pulses of 30 sec at 4°C) followed by a treatment with antarctic phosphatase and re-phosphorylated with polynucleotide kinase (PNK). Afterwards oligonucleotide adapters were ligated to the 5' and 3' ends of the RNA samples. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The resulting cDNAs were amplified with PCR using a high fidelity DNA polymerase. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). The cDNAs have a size range of 200-500 bp. The cDNA pool was either paired-end sequenced using 2x75 bp read length (Replica R2 samples) or single-end sequenced using 50 bp read length on an Illumina NextSeq 500 system. Adapter ligation on fragmented RNA samples.