GSM3883454: MS8345 PBT2 3; Escherichia coli; RNA-Seq
RNA was isolated using the RNeasy Plus Kit (Qiagen). Briefly, bacteria were grown to OD600 of 0.5 in CA-MHB in the presence or absence of PBT2. Two volumes of RNAprotect (Qiagen) were added to the cultures and the samples were then centrifuged at 5,000 x g for 25 min at 4°C to pellet cells. RNA was isolated from the dry pellet as per the manufacturer's instructions. To ensure complete removal of DNA, the RNA was then further purified using the TURBO DNA-free kit (Thermo Fisher Scientific) according to the manufacturer's instructions. RNASeq analysis was performed at the Australian Genome Research Facility. The cDNA library was prepared using a Ribo-zero stranded protocol. In brief, rRNA was depleted with Ribo Zero, RNA was fragmented (heat and divalent cations) and 1st strand cDNA synthesis was done with SuperScript II Reverse Transcriptase (Invitrogen). For the 2nd strand cDNA synthesis, the strand was “marked” with dUTP.  A 3' adenylation of DNA fragments was performed followed by sequencing adapter ligation (utilizing T-A pairing of adapter and DNA fragments). The library was amplified by PCR (amplification of “unmarked” 1st strand only). Libraries were assessed using either Agilent's Bioanalyzer DNA 1000 chip or TapeStation D1K TapeScreen system. qPCR was used to quantify individual libraries before normalizing (2 nM) and pooling. Libraries were pooled and clustered through the Illumina cBot system using TruSeq PE Cluster Kit v3 reagents followed by sequencing on the Illumina HiSeq 2500 system with TruSeq SBS Kit v3 reagents with 110 (101 read 1, 9 cycles index read). Libraries were sequenced with a HiSeq 2500 ultra-high-throughput sequencing system (Illumina) to produce 50-base-single end reads. An average of 30 million reads per sample were generated, uploaded to the Galaxy web platform (public server at usegalaxy.org), and mapped to the reference genome (K. pneumoniae MS6671 accession number: LN824133; E. coli accession number: CP025401.1) using bowtie2 with default parameters. Read counts were determined using featureCounts and differential gene expression was analyzed with edgeR with the CPM threshold set at 2.0. Figures were generated using ggplot2 and ggpubr packages in R-studio
GEO Accession
GSM3883454