RNA-seq analysis of Campylobacter jejuni NCTC 11168 fur perR mutant
RNA for RNA-seq analyses was extracted from a C. jejuni NCTC 11168 fur perR mutant, grown to an optical density of 0.4 at 600 nm (A600). Total RNA was purified omitting size selection, to avoid the loss of small RNA molecules. The exclusion of rRNA and tRNA was also omitted, to avoid the potential loss of other RNA species. RNA was isolated using hot phenol (Mattatall & Sanderson, 1996) to ensure that small RNAs would not be removed by the extraction procedure. The RNA was treated with DNase I to remove residual genomic DNA. The RNA-samples were also used to generate strand-inspecific RNA-seq libraries for Illumina sequencing, using instructions from the manufacturer (Illumina), and further analysed using an Illumina HiSeq2000 sequencer. The reads were aligned against the C. jejuni NCTC 11168 genome sequence using Segemehl version 0.0.9.3, and converted into number of reads per nucleotide position. Graphs representing the number of mapped reads per nucleotide were visualized using the Integrated Genome Browser software from Affymetrix. Transcript levels of individual genes were expressed as Reads Per Kilobase per Million mapped reads (RPKM) values, calculated after mapping of reads using CLC Genomics Workbench v5 (CLC Bio).
Institute of Food Research
Campy_fur_perR_RNAseq