Illumina MiSeq paired end sequencing; 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.
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.
The cells were harvested in the exponential growth phase and frozen in liquid nitrogen. Total RNA was isolated from 3 biological replicates of C. glutamicum 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).  cDNA library enriched for primary 5′-transcripts according to Peifer-Sancar et al. (2013): I) Depletion of stable RNAs using the Ribo-Zero rRNA removal kit II) RNA fragmentation by metal hydrolysis III)  Digestion of non native 5'-Mono/Di-phosphate ends by Terminator 5′-phosphate-dependent exonuclease IV)Conversion of 5′-triphosphates of primary transcript ends to monophosphates to enable adapter ligation V)Full RNA adapter ligation VI)cDNA synthesis by the usage of a loop adapter which hybridizes via a 3′-NNNNNNN-tail to the 3′-end of the RNA fragment VI)Amplification and Indexing by Adapter-specific primer