Determination of transcriptional start sites in the presence of GABA for Corynebacterium glutamicum

In this study, we analyzed the regulation of ?-aminobutyrate (GABA) utilization in Corynebacterium glutamicum by the PucR-type transcriptional regulator GabR and by alternative nitrogen and carbon sources. Overall design: RNAseq analysis was performed to determine transcriptional start sites of genes that are related to GABA utilization. Cappable-seq RNA treatment was done by vertis Biotechnologie AG (Freising-Weihenstephan, Germany): Capping of the 5' triphosphorylated RNA with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) (NEB) using the vaccinia capping enzyme (VCE) (NEB) for reversible binding of biotinylated RNA species to streptavidin Capturing of biotinylated RNA species on streptavidin beads and elution to obtain the 5' fragment of the primary transcripts The Cappable enriched RNA samples were poly(A)-tailed using poly(A) polymerase. In order to remove residual 5'P-ends, the RNAs were treated with Antarctic Phosphatase (NEB). Then, the 5'PPPcap structures were converted to 5'P using the RppH enzyme (NEB; analog to CAP-Clip Acid Pyrophosphatase). Afterwards, an RNA adapter was ligated to the newly formed 5'-monophosphate structures. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 10-20 ng/µl using a high fidelity DNA polymerase. For Illumina sequencing, 100 – 300 bp long 5' fragments were isolated from the full-length cDNAs. Forthis purpose the cDNA preparations were fragmented and the 5'-cDNA fragments were then bound to streptavidin magnetic beads. The bound cDNAs were blunted and the 3' Illumina sequencing adapter was ligated to the 3' ends of the cDNA fragments. The bead bound cDNAs were finally PCR-amplified. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 75 bp read length.