Valine-Induced Isoleucine Starvation in Escherichia coli K-12 Studied by Spike-In Normalized RNA Sequencing

Here we report on an RNAseq method in combination with spike-in cells to measure global changes in the transcription pattern after valine-induced isoleucine starvation of a standard E. coli K12 strain. Due to the spike-in method we were able to show that ribosomal RNA is degraded during isoleucine starvation and we showed how this change in cellular RNA content affect the estimated regulation of mRNA levels compared to if spike-in were not utilized. Our analysis also showed that induction of starvation by sudden addition of high valine concentrations provoked prominent regulatory responses outside of the expected ppGpp, RpoS and Lrp regulons Three samples were collected during steady-state growth immediately before starvation and five samples in total were collected in a time series; 5, 10, 20, 40 and 80 minutes after L-valine addition. In Addition, 4 rifampicin treated samples in total was collected after 80 minutes of Isoleucine starvation in a time series; 10, 15, 25 and 45 minutes after addition of rifampicin. All samples were spiked-in with a culture grown in a parallel, expressing Vibrio cholerae sRNA (qrr2) in high amounts. 1% spike-in culture were added to each sample based on sample OD