G.oxydans TSS data set with PrhaSR-PrhaBAD-mNG
For Cappable-seq RNA, the RNA sample was enriched by 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. Then, streptavidin beads were used to capture biotinylated RNA species followed by elution to obtain highly enriched 5' fragment of the primary transcripts. The Cappable enriched RNA sample was poly(A)-tailed using poly(A) polymerase. In order to remove residual 5'-P-ends, the RNA was treated with Antarctic Phosphatase (NEB). Then, the 5'-PPP cap structures were converted to 5-P using the RppH enzyme (NEB). 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 MMLV reverse transcriptase. The resulting cDNA was PCR-amplified to about 10-20 ng/L using a high fidelity DNA polymerase. For Illumina sequencing, 100300 bp long 5' fragments were isolated from the full-length cDNA. For this purpose the cDNA preparation was 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 library was sequenced on an Illumina NextSeq 500 system using 75 bp read length.