Instrument: Illumina MiSeq
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA was isolated from cells grown at exponential phase (OD600 = 0.5) using Trizol reagent (Invitrogen). The bacterial pellet obtained from 4 mL culture was resuspended in 1 mL Trizol, transferred into FastPrep Lysis Beads & Tube (MP Biomedicals) and mechanically lysed using beadbeater at a maximum speed for 20 sec 6 times. After adding 200 µL chloroform to the lysed cells followed by centrifugation at 12,000 x g for 15 min at 4 °C, the aqueous supernatant was taken and then precipitated using 500 µL isopropanol. Afterwards, crude RNA samples were treated with DNase I (Roche Diagnostics). After purification using phenol/chloroform/isoamyl alcohol (ratio 25:24:1), RNA was precipitated with ethanol. Purified total RNA pellets were dissolved in 50 µL RNase-free water. For whole transcriptome cDNA library preparations, 2 µg total RNA from C. diphtheriae NCTC 13129 and the ΔdtxR mutant were used. Stable RNAs were depleted with the RiboZero Kit according to manufacturer's instructions (Epicentre). Afterwards, the remaining mRNA was purified using RNA MinElute columns (Qiagen) and checked for quality with the Bioanalyzer (Agilent). Fragmentation of mRNA, reverse transcription to cDNA, adenylation of 3' ends, adapter ligation and PCR amplification were performed according to TruSeq Stranded mRNA library instructions (Illumina). Prior to paired-end sequencing of the whole transcriptome cDNA libraries on an Illumina MiSeq, the quality and concentration of the libraries were checked using the Bioanalyzer (Agilent). For the primary 5'-end cDNA library, 2× 5 µg wild type RNA was used. The preparation protocol has been described previously in detail [Pfeifer-Sancar2013]. In the present study, the experimental workflow was changed at three steps. Non-primary transcripts were digested with terminator exonuclease (Epicenre) at 30 °C for 60 min and subsequently at 42 °C for 30 min. The 5' adapter ligation was performed for 60 min at 30 °C with 1 µL 60 µM adapter. After cDNA amplification, the two libraries were purified and size-selected by gel electrophoresis for fragment sizes between 100 and 1,000 bp. The cutoff of 100 bp was chosen to reduce adapter dimers in the finished library. Due to the fact that the preparation workflow involves the use of two adapters, which together have a length of 66 nt, only transcripts smaller than 40 nt are not present in the final RNA-seq data. Sequencing was performed in single-read mode with 75 nt read length for the primary 5'-end cDNA library.