title: Illumina HiSeq 4000 paired end sequencing; RNA-seq of Escherichia coli MG1655 and Salmonella Typhimurium SL1344 wild-types and their respective AcrB D408A mutants in LB broth, in stationary phase
design: RNA-seq of Escherichia coli MG1655 and Salmonella Typhimurium SL1344 wild-types and their respective AcrB D408A mutants in LB broth, in stationary phase
library_construction_protocol: Cultures were plunged into 2/5 of ice-cold phenol:ethanol (5% phenol in 95% of pure ethanol). Samples were incubated on ice for 30 minutes and centrifuged at 3500 Xg for 20 minutes at 4C to concentrate the culture. Phenol:ethanol was removed and pellets were stored at -80C overnight before RNA extraction. Protocol from Kroger et al., 2013 doi: 10.1016/j.chom.2013.11.010 Overnight cultures of single colonies were diluted 1:100 in sterile LB broth. Cultures were incubated at 37C, shaking at 200rpm for 10 hours.  RNA was extracted from quenched bacterial pellets using RNeasy mini kit (QIAGEN, cat. number 74104). The manufacturer's instructions were followed. Quality of extracted RNA was assessed by Bioanalyzer and Nanodrop. Library was constructed by the Beijing Genomics Institute (BGI) as a contracted service.