RNA-seq analysis of E. coli transcriptome

MG1655/pBAD24 or CDS091 (MG1655 ΔphoB)/pBAD24 cells were grown at 37°C with aeration in MOPS minimal medium with 0.2 mM K₂PO₄, 0.4% glucose, and 100 μg/mL ampicillin to an OD₆₀₀ of 0.5 to 0.6. Arabinose was added to a final concentration of 0.2% for 7 min. Note that addition of arabinose is not expected to impact the expression of PhoB-regulated genes. RNA was isolated using a modified hot-phenol method, as previously described (76 (link)). Samples were treated with Turbo DNase (Ambion) to remove genomic DNA, rRNA was removed using the Ribo-Zero rRNA removal kit for Gram-negative bacteria (Epicentre/Illumina), and libraries were prepared with the ScriptSeq Complete kit for bacteria (Epicentre/Illumina) (76 (link)). Libraries were sequenced on a HiSeq 2000 (Illumina) by the University at Buffalo Next-Generation Sequencing Core Facility. RNA-seq data were aligned to the E. coli MG1655 genome (GenBank accession no. NC_000913.3) using BWA for Illumina (v0.5.9-r16) (78 (link)) on Galaxy (https://usegalaxy.org) (79 (link)). Read counting, normalization, and differential expression analysis were performed in R using GenomicAlignments (v1.28) summarizeOverlaps (80 (link)) and DEseq2 (v1.32; betaPrior = FALSE) (81 (link)).