Illumina MiSeq paired end sequencing; 5'-RNA-seq of 5'-enriched primary coding RNA in Staphylococcus aureus wildtype to detect Transcription Start Sites
5'-RNA-seq of 5'-enriched primary coding RNA in Staphylococcus aureus wildtype to detect Transcription Start Sites
S. aureus cells of 3 replicate experiments cultivated in Luria Bertani (LB) medium at 37°C until cells have reached an optical density at 540 nm of 2.0 were harvested and disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 Ribolyzer. RNA isolation was performed using the phenol-chloroform-isoamylalcohol approach. Cultivation was performed in Luria Bertani (LB) medium in triplicate at 37°C until cells have reached an optical density at 540 nm of 2.0. Cells were harvested by centrifugation, washed with Belitsky minimal medium (BMM) and adapted to BMM for one hour.  Cells were disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 Ribolyzer. RNA isolation was performed using the phenol-chloroform-isoamylalcohol approach. After precipitation with 3 M sodium acetate and isopropanol, the total RNA was washed with cold ethanol and the pellet was solved in sterile water. Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. 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
Experimental Factor: individual
mixed pool of 3 replicates