Illumina MiSeq paired end sequencing; Studying the phosphosugar stress in Bacillus subtilis using differential gene expression analysis of wildtype and manA deletion mutant grown on LB mannose medium
Studying the phosphosugar stress in Bacillus subtilis using differential gene expression analysis of wildtype and manA deletion mutant grown on LB mannose medium
B. subtilis 168 strains KM0 and KM642 were cultured in LB with 1% mannose and the cell pellets were isolated after 3.5 h of incubation at 37 °C for transcriptome analysis by RNA-Seq RNA isolation was performed on sequential biomass samples (approximately 2 ∙ 10^9 cells), harvested in a fast sampling procedure, basically using the RNeasy Mini Kit (Qiagen) including a double DNA digestion step. RNA quality and quantity was checked by using Trinean Xpose system (Gentbrugge,Belgium) and by Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies). RIN > 9. PCR was performed to assure no DNA remained in the samples. 2.5 µg of total RNA with RNA integrity number (RIN) > 9.0 were used for cDNA lbrary preparation. Stable RNAs were depleted with the Ribo-Zero rRNA Removal Kit (Bacteria) according to manufacturer's instructions (Illumina). Afterwards the remaining mRNA was purified using RNA MinElute columns (Qiagen) and checked for quality and successful rRNA depletion with the Agilent RNA Pico 6000 kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). The TruSeq Stranded mRNA Library Prep Kit from Illumina was applied to prepare the cDNA libraries.
Experimental Factor: strain
KM642