Illumina HiSeq 2500 paired end sequencing; RNAseq of uropathogenic E. coli strain EO399, grown in conditions resembling passage through the gut; coli was grown in the presence or absence of 1 of 3 organic acids (acetic, butyric or propionic) at pH5.5 or pH7 and in aerobic or anaerobic conditions
RNAseq of uropathogenic E. coli strain EO399, grown in conditions resembling passage through the gut; coli was grown in the presence or absence of 1 of 3 organic acids (acetic, butyric or propionic) at pH5.5 or pH7 and in aerobic or anaerobic conditions
E. coli cultures were grown overnight, each inoculated from a single colony grown in M9supp Growth was monitored spectroscopically until an OD600 of 1.8 was reached.  A dilution and replenishment protocol was then applied to achieve steady state growth as described previously [37], by removing 5 ml of culture and replacing it with 5 ml of pre-warmed M9supp.  This was done every 30 minutes for an hour for the anaerobic cultures, and every fifteen minutes for the aerobic cultures; pilot experiments established that this maintained the culture density between an OD600 of 1.8 and 2 (data not shown).  To impose the stress, 5 ml of culture was then removed, and 5 ml of pre-warmed M9supp at the appropriate pH with or without additional SCFA were added to create the specific stress condition.  Growth was then continued for 15 minutes.  Five ml of culture were removed from each flask and transferred to an equal amount of Qiagen RNA Protect and mixed thoroughly. Cells were pelleted by spinning at 4000rpm for 5 minutes in a chilled centrifuge, and pellets were stored at -80 C for downstream RNA purification. RNA was purified from the pellets collected as above using Qiagen RNeasy kits according to manufacturer's instructions.  The optional on-column DNA digestion step was done for all samples to ensure there was no DNA contamination of samples. PCR amplification of the evgS gene was used to confirm the absence of genomic DNA (data not shown). All samples were checked for quality using a Prokaryote Total RNA Nanochip, and analysed using the Agilent Bioanalyser in order to provide a RNA integrity number (RIN). Only samples with a RIN of 7 or higher were used for sequencing. Ribo-depletion was done using the Illumina Ribo-zero rRNA removal kit according to manufacturer's instructions.