GSM3462628: M418; Pseudomonas aeruginosa; RNA-Seq
Total RNA was extracted by the hot phenol method using the TRI® Reagent protocol (Ambion) and subjected to two DNase I treatments with the Turbo DNA-free kit (Ambion) and RNaseOUT (Invitrogen). RNA quality, including purity, integrity and yield, was assessed by electrophoresis of 1 µg of total RNA and by UV absorption at 260 nm in a ND-1000 spectrophotometer (NanoDrop Technologies, USA). The rRNA from 4 µg of total RNA was removed using the Ribo-Zero rRNA removal kit (Illumina) following the manufactures' recommendations. Sequencing libraries were prepared with the NEBNext Ultra directional RNA library Prep kit (New England Biolabs). RNA was fragmented at 94 °C for 7.5 min and first strand cDNA synthesis was performed at 42 °C during 50 min using the adaptor and primers recommended by the manufacturer (NEBNext Multiplex Oligos, Illumina).