GSM4633759: control_B+TEX; Pseudomonas aeruginosa PAO1; RNA-Seq
Flash frozen samples were used for RNA using the Hot phenol protocol (described e.g. in PMID: 31796567) Total RNA extracted from ɸKZ-infected and uninfected P. aeruginosa PAO1 cells was divided in two pools, named TEX- and TEX+. Samples were examined by capillary electrophoresis and fragmented using ultrasound (4 pulses of 30 sec at 4 °C), followed by T4 Polynucleotide Kinase (NEB) treatment. TEX+ samples were subjected to Terminator-5´-phosphate-dependent exonuclease to enrich primary transcripts whereas TEX- samples remained untreated. For cDNA synthesis RNA samples were poly(A)-tailed and remaining 5´PPP structures were removed. After ligation of an RNA adapter to the 5´P end, first-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. PCR amplification of cDNAs was conducted using a high-fidelity DNA polymerase and cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). Quality control was carried out by capillary electrophoresis. All described procedures were performed by Vertis Biotechnology (Germany). TruSeq Stranded Library Preparation Kit (Illumina)