title: Illumina HiSeq 2000 sequencing; Transcriptional response of Pseudomonas aeruginosa to treatment with the antibacterial compound SPI031
design: Transcriptional response of Pseudomonas aeruginosa to treatment with the antibacterial compound SPI031
library_construction_protocol: Overnight cultures of P. aeruginosa were diluted 1/100 in 1/20 TSB and were grown for 3 h until late-exponential phase. Cells were treated for 5 min with 0.2x MIC of SPI031 The RNA content of 20 mL bacterial culture was stabilized by adding 1:5 volume of ice-cold phenol:ethanol (5:95). Cells were flash frozen in liquid nitrogen and stored at -80 °C. For RNA isolation, cell pellets were resuspended in TRIzol and cells were lysed by mechanical disruption using a Precellys 24 (Bertin Technologies) at 6500 rpm for 45 seconds with 0.25 ml of 0.1 mm glass beads. Next, organic and aqueous phases were separated by Phase Lock Gel tubes (heavy type) and total RNA was isolated using the Pure Link RNA mini Kit (Ambion). RNA samples were DNase treated using Turbo DNase-free kit (Ambion) according to the manufacturer’s protocol. To increase yields, RNA was precipitated in 3 volumes of isopropanol and 1/10 volume of sodium acetate (3 M, pH 5.2), washed twice in ethanol and resuspended in nuclease-free ultrapure water. RNA integrity was analyzed using Experion RNA StdSens Chips (Bio-Rad). RNA quantity and purity was assessed using a NanoDrop ND-1000 spectrophotometer. RNA was subjected to ribosomal RNA depletion using the Ribo-Zero Magnetic Kit for gram negative bacteria (Epicentre). Libraries for RNA Seq were constructed from the rRNA-depleted samples using standard Illumina protocols.