5'-specific RNA-Seq of Corynebacterium glutamicum wildtype, deletion mutant sigE and deletion mutant cseE reveals TSS and promoters depending on SigE and thereby overlapping SigH and SigE sigma factor regulons.

Total RNA was isolated from 3 biological replicates of C. glutamicum wildtype, deletion mutant sigE and deletion mutant cseE cells by a Quick-RNA Miniprep Plus kit (Zymo Research). The samples were treated with DNase (Roche Diagnostics) and RNA was purified with an RNA Clean&Concentrator-5 kit (Zymo Research). A primary 5'-end-specific cDNA library was constructed for sequencing with the aim of defining TSSs using the protocol described previously (Albersmeier et al., 2017; Wittchen et al., 2018). Briefly, rRNA was depleted and the RNA samples were treated with a terminator 5'-phosphate-dependent exonuclease (Illumina) to remove non-primary transcripts. The primary 5'-triphosphate ends of the primary transcripts were treated with RNA 5'-polyphosphatase to convert them into 5'-mono phosphate ends, and the 5'-adapter was ligated to the produced 5'-ends. Then, reverse transcription with a stem-loop DNA adapter was carried out and the library was amplified. The primary 5'-end cDNA library was then purified and size-selected for fragments approximately 100-1000 nt in size by gel electrophoresis and quantified. The fragments were finally sequenced with an Illumina MiSeq.