GSM1197183: Campylobacter jejuni wildtype dRNA-seq, noTEX; Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819; RNA-Seq
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 sample was split in two equal fractions, and one of the two fractions was treated with Terminator Exonuclease (TEX, Peicentre Biotechnology) for enrichment of primary RNAs (Sharma et al., 2010). Both samples were treated with Tobacco Acid Phosphatase (TAP, Cambio, UK) to generate 5'-P ends for downstream ligation of 454 adapters. 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. The cDNA fragments were barcoded 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 FLX/Titanium sequencer using standard protocols from the manufacturer.
GEO Accession
GSM1197183