RNA-seq analysis of ygfB deletion mutant

The strains ID40 and ID40∆ygfB were used to perform RNA sequencing and differential gene expression analysis (ID40 vs ID40∆ygfB). The strains were subcultivated for 3 h in 5 ml LB medium. A total of four independent replicates per strain were used in the sequencing and analysis. RNA was isolated using the Quick-RNA™Fungal/Bacterial MiniprepKit (Zymo Research) according to manufacturer´s instruction. Subsequently, 15 µg RNA in 50 µl water was digested with 10 U DNAse I (Roche). The quality of the RNA was controlled by determination of the RNA Integrity Index using Agilent BioAnalyzer High Sensitivity DNA Assays. Successful depletion of DNA was controlled by qPCR and RT-qPCR for the rpoS and the ygfB genes. Next, the Zymo-Seq RiboFree total RNA Library Prep Kit (Zymo Research) was used to deplete ribosomal RNA and prepare samples for sequencing. For this step, 2 µg RNA per sample were used. Sequencing was performed with Illumina NextSeq500 (2×75 bp, MidOutput Flowcell). Mapping of sequencing reads and counting was performed using the subread package in R and the ID40 genome as a reference (https://www.ebi.ac.uk/ena/browser/view/LR700248)^{37 (link)}. Differential gene expression analysis was performed using DeSeq2^{38 (link)}.