GSM4826432: ∆yebEFG ESP nalidixic acid Rep1; Salmonella enterica; RNA-Seq
Total RNA was isolated using the Direct-zol RNA MiniPrep Plus kit (Zymo Research). RNA quality and concentration was determined by nanodrop. cDNA libraries were prepared and sequenced by Vertis Biotechnologie AG (Freising, Germany). Ribodepleted RNA samples were fragmented by ultrasound pulses (4 pulses of 30s each at 4ºC). Oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was amplified to about 10-20 ng/ml using a high fidelity DNA polymerase. The cDNA was amplified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis. cDNA samples were pooled in equimolar amounts, size-selected to 200-550 bp and sequenced on an Illumina NextSeq 500 system using 75 bp read length.