GSM5018542: femI-KO +Mycobactin rep2; Pseudomonas aeruginosa; RNA-Seq
Strains were grown overnight at 37 °C followed by sub-culturing into fresh medium and grown to mid-exponential phase. Total RNA was extracted by TRIzol-based method (Life Technologies, CA, USA). In brief, the cultures were centrifuged at 12, 000 g for 5 minutes. The supernatant was discarded, cell pellets were resuspended in 1 ml of TRIzol and then incubated at room temperature for 5 minutes to permit complete dissociation of nucleoproteins complex. Followed by adding 0.2 ml chloroform into the tube and mix well, the samples were centrifuged (12, 000 g at 4 °C for 15 minutes) to form three layers. The upper aqueous phase was transferred to a fresh tube and added by 0.5 ml cold isopropanol to precipitate RNA. After centrifugation, the supernatant was removed, and the pellet was suspended in 1 ml 75% ethanol. Samples were centrifuged and the supernatant was discarded, air-dried RNA pellet was resolved in 50 µL RNase-free Water. RNA integrity was measured using Bioanalyzer 2100 (Agilent, Santa Clara, CA) and rRNA was removed from 1 mg of total RNA with Ribo-Zero Magnetic Gold Kit (Epicentre Biotechnologies, Madison, WI, USA). To construct the RNA-Seq library, TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) was used. rRNA-depleted RNA was fragmented into small pieces using Elute Prime Fragment Mix. First-strand cDNA was synthesized with First Strand Master Mix and Super Script II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Following purification by Agencourt RNAClean XP beads (Beckman Coulter, CA, USA), the second-strand cDNA library was synthesized using Second Strand Master Mix and dATP, dGTP, dCTP, dUTP mix. Purified fragmented cDNA was end repaired (30 minutes at 37 °C) prior to ligating sequencing adapters. Amplified RNA-Seq libraries were purified by using AMPureXP Beads.