4.10. Total RNA Isolation and Sequencing

Enriched cultures were re-grown in media containing 1.4 µM TMP, 49.3 µM TCATa, 98.5 µM TCATd or without addition of compounds for the control, respectively, for 6 h, after which the cells were harvested by centrifugation. RNA was isolated using a High Pure RNA Isolation Kit (Roche, Basel, Switzerland).

RNA samples were sequenced at BGI (Wuhan, China), who performed rRNA removal and library preparation. Transcriptome sequencing was performed on DNBseq™. Raw sequence reads were analyzed for quality and mapped to E. coli K12 (ASM983882) using Bowtie2 [27]. Count values were used as an input for the T-REx analysis pipeline for statistical analysis to determine differentially expressed genes [28]. For the T-REx analysis, a text file describing the factors, contrasts and classes specifying genes were written. TopHits were further evaluated with GSEA- Pro v. 3 [29]. Euler diagrams were made with RStudio version 1.3.1093. The RNA-seq data were uploaded under GEO accession number: GSE169069.