title: Differential RNA-seq of Campylobacter jejuni NCTC 11168 fur perR mutant, late log growth phase
design: RNA was isolated from the fur perR mutant of C. jejuni strain NCTC 11168 (Palyada et al, 2009), grown to late log phase. 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, followed by optional treatment with Terminator Exonuclease (TEX, Peicentre Biotechnology) for enrichment of primary RNAs (Sharma et al., 2010; Zhang et al., 2008), and treatment with Tobacco Acid Phosphatase (TAP, Cambio, UK) to generate 5'-P ends for downstream ligation of 454 adapters (Sharma et al., 2010). After ligation of an RNA oligonucleotide to the phosphorylated 5’-ends of RNA, and polyadenylation of RNA, first strand cDNA was generated using an oligo-dT containing 454-B primer (Table S13). The cDNA fragments were barcoded (ACGTGC for TEX-treated RNA, AGCGTA for non-treated RNA) and amplified, and used for generation of cDNA libraries for the 454 FLX system at Vertis Biotech, Germany. These libraries were subsequently analysed using a Roche Titanium FLX sequencer at The Genome Analysis Centre, Norwich, UK, as previously described (Sharma et al., 2010). The enrichment procedure significantly reduced the level of 23S and 16S rRNA, but led to an increase in 5S rRNA, while tRNA levels were not altered (Sharma et al., 2010).     Mapping of 454 reads and annotation of transcription start sites  Sequencing reads were grouped based on the barcode tag, the 5' adapter was clipped, and reads of >70% A were removed. The remaining reads were aligned against the C. jejuni genome NCTC 11168 genome sequence using Segemehl version 0.0.9.3 (Hoffmann et al., 2009), 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 (Nicol et al., 2009; Sharma et al., 2010). TSS were manually annotated based on a higher and characteristic cDNA coverage of the 5'-end of a given cDNA in the library constructed with terminator exonuclease-treated RNA. Genomes were annotated and analysed using Artemis (Carver et al., 2008).    - Carver T, et al (2008) Bioinformatics 24, 2672-2676.  - Hoffmann S, et al (2009) PLoS Comput Biol 5, e1000502.  - Mattatall NR & Sanderson KE (1996) J Bacteriol 178, 2272-2278.  - Nicol JW, et al (2009) Bioinformatics 25, 2730-2731.  - Palyada K, et al (2009) BMC Genomics 10, 481.  - Sharma CM, et al (2010) Nature 464, 250-255.  - Zhang H, et al (2008) PLoS Pathog 4, e1000219.