title: GSM3501781: P. aeruginosa_vehicle (EtOH)_mRNA (duplicate); Pseudomonas aeruginosa; RNA-Seq
library_construction_protocol: Bacterial cells were recovered and used to isolate total RNA using a QIAGEN RNeasy Plus Mini Kit (cat. no. 74134). Concentration and purity of RNA samples were determined by measuring the absorbance (A260:A280) using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). Total RNA samples were then processed for the depletion of ribosomal RNA (rRNA) following the manufacturer's user manual for Ribo-Zero rRNA removal kit for bacteria from Illumina (Document # 15066012 V02). RNA libraries were prepared following the Lexogen's user manual of Sense Total RNA-seq library preparation kit (cat.no. 009.24, 020.24, 022.24). Briefly, 10 ng of rRNA-depleted RNA was used as the starting material for the generation of libraries. The library generation started with the random hybridization of the starter/stopper heterodimer mix to the rRNA-depleted RNA. These starter/stopper heterodimers contained Illumina-compatible linker sequences. A single-tube reverse transcription and ligation reaction was then performed to extend the starter to the next hybridized heterodimer, where the newly synthesized cDNA insert was ligated to the stopper. The second strand synthesis was then performed to hydrolyze the RNA and then the library was converted to double-stranded DNA, which was purified using magnetic beads to adjust the library length and to ensure the complete removal of second strand synthesis reaction components. The library amplification was then performed to add the complete adaptor sequences required for cluster generation and to produce sufficient material for quality control and sequencing; i7 indices were added during this step in order to uniquely multiplex the samples for the sequencing run. The resulting RNA libraries were then purified using magnetic beads to get rid of PCR components, which could interfere with quantification and sequenced on an Illumina MiSeq instrument.
attributes.tag: GEO Accession
attributes.value: GSM3501781