E-MTAB-7385 - 5'-RNA-seq of 5'-enriched primary coding RNA in Staphylococcus aureus wildtype to detect Transcription Start Sites
Submitted on 6 December 2017, last updated on 8 November 2018, released on 1 April 2019
Staphylococcus aureus subsp. aureus
To detect Transcription Start sites (TSS) in Staphylococcus aureus wildtype a cDNA library enriched for primary 5′-transcripts according to Peifer-Sancar et al. (2013) was prepared and sequenced on Illumina MiSeq system to retrieve 5′-ends of primary transcripts. 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. S. aureus cells of 3 replicate experiments 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. 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. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. The method from Peifer-Sancar et al. (2013) was used to prepare a cDNA library enriched for primary 5′-transcript. The resulting cDNAs were sequenced paired end on an Illumina MiSeq system (San Diego, CA, USA) using 75 bp read length.
RNA-seq of coding RNA, strain or line design