title: RNA-seq of PAO1 without Ca, biological replicate 1
design: Total RNA was isolated from P. aeruginosa mid-log cultures grown in BMM with no added or 5 mM Ca2+ by using RNeasy Protect Bacteria Mini kit (Qiagen) or ZR Fungal/ Bacterial RNA MiniPrepTM (Zymo Research). The quality of the purified RNA was assessed by Bioanalyzer 2100 (Agilent) and 1% agarose gel electrophoresis. RNA-Seq analysis was performed at Vertis Biotechnology AG, Germany using differential RNA-seq (dRNA-seq). The capable RNA library was generated by tagging the 5 triphosphophate with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) (NEB: N0761S) by using the vaccinia capping enzyme (VCE) (NEB: M2080S). The biotinylated RNA was captured on streptavidin beads and the 5 triphosphate was converted to monophosphate. The RNA was then ligated onto an adaptor, and the library was amplified and sequenced on an Illumina NextSeq 500 system using 75 bp read length. Sequence analyses of such library enables identification of transcriptional starts at the single base resolution.