title: yqjD_A020
organism: Escherichia coli str. K-12 substr. MG1655
description: Protocols: Strains were constructed by mating conjugal plasmids from the mobile-plasmid collection (Saka et al 2005) into E. coli K-12 substrain MG1655. Strains were grown and biological material was collected as described in “Growth” protocol. Strains were grown to saturation in M9 glucose minimal medium, diluted 1:100 into M9 glycerol minimal medium and grown for 9 hrs (early exponential phase). IPTG (100 mM) was added to induce DDP overproduction and cultures were grown for an additional 8 hrs. Total RNA was isolated from 1 ml of culture (~10^8 cells) for each of two biological replicates using the RNeasy Mini Kit (Qiagen), according to the manufacturer's protocol. RNAprotect Bacterial Reagent (Qiagen) was used to stabilize RNA during harvest and enzymatic cell lysis. After elution, total RNA was treated with RNase-free DNase I (NEB), according to the manufacturer's protocol. RNA was recovered by phenol-chloroform extraction and ethanol precipitation. Ribosomal RNA was depleted using RiboZero (Epicentre/Illumina), according to the manufacturer's protocol. Remaining RNA was concentrated by ethanol precipitation. Approximately 100 ng of rRNA-depleted RNA was used to construct libraries using the TruSeq Stranded mRNA Library Preparation Kit (Illumina). Libraries were prepared according to the manufacturer's protocol, using recommended modifications for previously isolated mRNA (poly-A RNA enrichment steps excluded). Final RNA-seq libraries were run on a BioAnalyzer (Agilent) to estimate the average fragment size (~800 bp) and the concentration of adapter-ligated library fragments was determined using the qPCR-based Illumina Library Quantification Kit (KAPA Biosystems).