title: Illumina MiSeq paired end sequencing; RNAseq of Bacillus subtilis wildtype and cspB-cspD deletion mutant reveals that the lack of the cold shock proteins CspB and CspD affects the expression of about 20% of all genes
design: RNAseq of Bacillus subtilis wildtype and cspB-cspD deletion mutant reveals that the lack of the cold shock proteins CspB and CspD affects the expression of about 20% of all genes
library_construction_protocol: B. subtilis 168 wild type and the cspB cspD double mutant GP1971 were grown in LB medium to an OD600 of 0.2 and were harvested by mixing them 5:3 with frozen killing buffer (20 mM Tris-HCl pH 7.5, 6 mM MgCl2, 20 mM NaN3) followed by snap-freezing of the pellets in liquid nitrogen.  B. subtilis 168 wild type and the cspB cspD double mutant GP1971 were grown in LB medium to an OD600 of 0.2. Total RNA was isolated by acid-phenol extraction. The RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 kit using an Agilent 2100 Bioanalyzer (Agilent Tech-nologies, Böblingen, Germany). Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA) was used to remove the rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina was applied to prepare the cDNA libraries.