In addition, we show the power of sequencing cDNA sequences for expression analysis by performing real-time PCR experiments for transcripts with high, medium and low R-value.
It is also important to recognize that experiments for studying transcript production are typically designed to look at single, specific transcripts originating from known promoters and often require production of a full-length transcript leading to generation of a measurable reporter protein.
The observation of increased expression of RNAIII, hla and spa in MRSA252 in response to LC-uFFAs is significantly different to previously published experiments for these transcripts in alternative strains [34], [35].
Nevertheless, before embarking on follow-up experiments for individual transcripts, validation experiments to map miRNA binding sites in the targets' 3′ UTRs and to identify the responsible miRNAs will be necessary.
In addition, directional RNA-seq data provide superior information compared to 5' RACE and primer extension experiments for mapping RNA transcripts and identifying potential promoter regions.
For transcript stability experiments, dghRFCMyc−his10/R5 cells were treated with 10 μg/ml actinomycin D and RNAs were prepared at 2 h intervals over 10 h.
For transcript profiling experiments, P. aeruginosa strains were grown in 250 ml flasks containing 50 ml LB buffered with 50 mM 3- N-morpholino) propanesulphonic acid, pH 7.0, at 3- N-morpholinoous shaking.
First we downloaded the set of human protein interactions provided by the PINA2 website [ 56] and extracted the network corresponding to proteins encoded by transcripts measured in our MDA-MB-468 experiments (transcripts for which we have no data were excluded from the network).
A number of such genes and regions as shown in Figure 2B typically occur in every RNA-Seq experiment and present the challenging cases for transcript level analysis.
Since the absence of Xrn1 leads to the accumulation of decapped RNAs (Hsu and Stevens, 1993), we could not use the TSS sequencing methodology applied for the other mutants and we thus used, for this particular experiment, a classical RNA sequencing approach for transcript quantification (see 'Materials and methods').
