description: Protocols: 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.
attributes.tag: ENA first public
attributes.value: 2021-03-01
attributes.tag: ENA last update
attributes.value: 2020-11-11
attributes.tag: External Id
attributes.value: SAMEA7566596
attributes.tag: INSDC center alias
attributes.value: University of Liverpool and University of Birmingham
attributes.tag: INSDC center name
attributes.value: University of Liverpool and University of Birmingham
attributes.tag: INSDC first public
attributes.value: 2021-03-01T12:18:13Z
attributes.tag: INSDC last update
attributes.value: 2020-11-11T11:56:08Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9762:Lund_Lab_Nov2020_Acetic_pH7_Anaero_pool
attributes.tag: aerobicity
attributes.value: anaerobic
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: labels
attributes.value: T15_acetate_pH7_AN_pool
attributes.tag: ph
attributes.value: pH7
attributes.tag: sample name
attributes.value: E-MTAB-9762:Lund_Lab_Nov2020_Acetic_pH7_Anaero_pool
attributes.tag: stimulus
attributes.value: acetic acid
attributes.tag: strain
attributes.value: EO399