GSM5035481: C. diphtheriae Wildtype Rep 2; Corynebacterium diphtheriae; RNA-Seq
C. diphtheriae RNA was prepared using RNeasy Mini Kits and RNase-free DNase Sets according to the manufacture's protocol (Qiagen). Briefly, cell pellets harvested from 3 ml log-phase cultures, cultivated in triplicates per condition, were suspended into 200 μl of chilled 10 mM RNA-free Tris-HCl, pH 8.0.  The suspension was added to a fast-protein tube (Q BIOgene) containing 700 μl RLT buffer (RNeasy Mini Kit, Qiagen), and cells were lysed using a Ribolyser (Hybaid). After centrifugation at 13,000 x g, RNA was purified from the supernatants accordingly. Purified RNA was treated by DNase (Qiagen) and subsequently cleaned by an RNeasy clean up kit (Qiagen). RNA quality was determined by an Agilent 2100 Bioanalyzer (Agilent Technologies) with an Agilent RNA Pico 6000 kit. RNA samples with the RNA integrity number (RIN) values of > 8.0 were stored at -800C for prior to RNA-seq analysis. 2 µg total RNA from C. diphtheriae NCTC 13129 and the delta-rnj2 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 TrueSeq 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).
GEO Accession
GSM5035481