RNA-Seq of Citrobacter rodentium
Total RNA was prepared from three replicates of pCitro-15a electroporated into E. coli EPI300(dap-) and C. rodentium. A single colony from each transformation was grown overnight to saturation under selection. Saturated cultures were diluted 1:50 into non-selective LSLB, and LSLB supplemented with 60 g/mL DAP for C. rodentium and EPI300 respectively. Cultures were grown for 2 hours at 37 C with 225 RPM shaking, reaching OD600 between 0.4-0.6.1.5 mL of culture were centrifuged was centrifuged at 16000 RCF for 5 minutes at 4 C. The supernatant was aspirated and the pellet was resuspended in 1 mL Trizol reagent (Invitrogen). Samples were incubated at 65 C for 10 minutes, and 0.2 mL of chloroform was addedand mixed by inverting for 15 seconds. Samples were incubated at room temperature for 3minutes before being centrifuged at 16000 RCF for 5 minutes at 4 C. The aqueous phase was transferred to a clean tube and 1 volume of ethanol was added to each. An NEB Monarch cleanup kit was used by adding samples to an RNA cleanup column and centrifuging at 11000RCF for 1 minute at room temperature. The column was washed twice by adding 500 Lof RNA cleanup wash buffer and centrifuging at 11000 RCF. The column was spun for an additional 1 minute at 11000 RCF to dry before eluted into 50 L nuclease-free water by centrifuging at 11000 RCF for 1 minute.