GSM4296999: dksA::cat+ greA::kan+ before stress; Escherichia coli; RNA-Seq
RNA was purified from 2 ml of cells harvested immediately before and 5 min after stress using the RiboPure™-Bacteria Kit (Ambion), according to the manufacturer's instructions. mRNA-sequencing was performed at the UAB Heflin Center for Genomic Sciences, using an Illumina NextSeq 500 as described by the manufacturer. Briefly, the quality of the total RNA was assessed using the Agilent 2100 Bioanalyzer. RNA with a RNA Integrity Number (RIN) of 7.0 or above was used for sequencing library preparation. We used the Agilent SureSelect Strand Specific mRNA library kit as per the manufacturer's instructions (Agilent). Library construction began with ribosome reduction with the RiboMinus protocol for Gram-negative and Gram-positive bacteria as described by the manufacturer (Life Technologies). The resulting mRNA was randomly fragmented with cations and heat, followed by first strand synthesis using random primers with inclusion of Actinomycin D (2.4 ng µl-1 final concentration). Second strand cDNA production was done with standard techniques, the ends of the resulting cDNA were made blunt, A-tailed, and adaptors were ligated for amplification and indexing to allow for multiplexing during sequencing. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for Illumina library quantitation (Kapa Biosystems) prior to cluster generation. Cluster generation was performed according to the manufacturers recommendations for onboard clustering (Illumina). We generated approximately 20 million single end 75 bp reads per sample.