RNA-Seq of E. coli under stress conditions

Escherichia coli strains and plasmids used in this study are listed in Supplementary Table S1. Escherichia coli strain MG1655 was grown in LB medium to OD₆₀₀∼0.1 at 37°C and exposed to 0.5% α-methylglucoside (αMG) for 15 min. Escherichia coli strain CV104 harboring either pHDB3 or pLCV1 plasmids was grown in morpholinepropanesulfonic acid (MOPS) minimal medium supplemented with 0.2% D-glucose to OD₆₀₀∼0.5 and exposed to 0.1 mM IPTG. Total RNA was extracted using the hot phenol method as described previously (17 (link)). RNA was treated with TURBO™ DNase (Ambion) according to the manufacturer’s protocol and resolved by electrophoresis on a 1.2% agarose gel to confirm integrity. Library construction and sequencing on the Illumina HiSeq2000 was performed at the W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign. Ribo-Zero rRNA Removal Meta-Bacteria Kit (Epicentre Biotechnologies) was used to remove rRNA from 1 µg of total RNA. The mRNA-enriched fraction was converted to indexed RNAseq libraries with the ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre Biotechnologies). The libraries were pooled in equimolar concentration and quantitated by quantitative PCR (qPCR) with the Library Quantification kit Illumina compatible (Kapa Biosystems). The pooled libraries were sequenced for 101 cycles plus 7 cycles for the index read on a HiSeq2000 using TruSeq SBS version 3 reagents. The fastq files were generated with Casava 1.8.2 (Illumina).