GSM4228161: C3719 OD3 replicate2; Pseudomonas aeruginosa; RNA-Seq
For bacteria RNA extraction we used the QIAzol lysis reagent and protocol (Qiagen). The quantity of the bacterial RNA samples was measured on a NanoDrop ND1000 system and their quality analyzed on the Agilent 2100 Bioanalyzer system with the Agilent RNA 6000 Nano kit protocol (Agilent Technologies), according to manufacturer's instructions. Bacterial RNA samples with RNA Integrity Number (RIN) > 7 were chosen for mRNA enrichment. The MICROBExpress™ Bacterial mRNA Enrichment Kit protocol (ThermoFisher Scientific) was performed on 5 – 10μg of bacterial RNA, according to the manufacturer's instructions. The quality and quantity of enriched mRNA were measured using the NanoDrop and the Bioanalyzer systems. After the assessment of the enriched bacterial mRNAs, libraries were prepared using the Ion Total RNA-Seq Kit v2 protocol and reagents (ThermoFisher Scientific) according to the manufacturer's instructions. The enriched mRNAs were fragmented, reverse transcribed and amplified after the addition of a specific (~10bp) barcode to each sample. The quantity and quality of the prepared libraries were assessed on a Bioanalyzer using the DNA High Sensitivity Kit, according to the manufacturer's instructions. All libraries were diluted to 1nM and subsequently pooled together in pools of eight. 40pM of pooled libraries were further processed for templating and enrichment, and loaded onto the Ion Proton PI™ V2 chips (ThermoFisher Scientific). The procedure was performed on the Ion Chef™ System using the Ion PI™ IC200 Chef kit, protocol and reagents (ThermoFisher Scientific), according to the manufacturer's instructions.
GEO Accession
GSM4228161