GSM2758955: RNA pool; Acinetobacter baumannii; RNA-Seq
Total RNA was isolated using TRIzol (Invitrogen) and contaminating DNA digested with DNase I cDNA libraries of the three ESP biological replicates were constructed using the NEBNext™ Ultra RNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s instructions. cDNA libraries of the RNA pool and TEX_RNA pool samples were constructed by vertis Biotechnologie AG (Freising) using an in-house protocol. Total RNA (DNase I digested) was fragmented by ultrasound (4 pulses of 30s at 4°C). The to construct dRNA-seq libraries, one part of the fragmented RNA was first treated with T4 Polynucleotide Kinase (NEB). RNA fragments with monophosphorylated 5' ends were then degraded using Terminator Exonuclease (TEX, Epicentre). The remaining 5'PPP structures were finally converted into 5' monophosphate ends using RNA 5' Polyphosphatase (5'PP, Epicentre). The other part of the fragmented RNA was dephosphorylated with Antarctic Phosphatase (NEB) and re-phosphorylated with T4 Polynucleotide Kinase. 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 number of cycles and the barcode sequences which are part to the 3' sequencing adapters. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. For Illumina sequencing, the samples were pooled in approximately equimolar amounts. An aliquot of the pool was analyzed by capillary electrophoresis.
GEO Accession
GSM2758955