Computational protocol: A broad range quorum sensing inhibitor working through sRNA inhibition

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Protocol publication

[…] cDNA was made from 1 μg of RNA using high-capacity RNA-to-DNA master mix (Applied Biosystems). Amplification was performed with SYBR green master mix in a Step One Plus thermal cycler (Applied Biosystems). The primers were designed using Primer Express software. Forty cycles were run with denaturation at 95 °C for 15 s, annealing at 55 °C for 30 s, and extension at 60 °C for 45 s. The gene rpoD was used as a control. See Table  for primer sequences. [...] RNA was purified according the descriptions above. Total RNA was checked for purity and intactness and sent for RNA sequencing at Beijing Genomic Institute. The resulting sequence data was analyzed in CLCbio’s Genomics Workbench v. 6.0.1 (QIAGEN). The reads were mapped back on the ≈3000 ORFs on the S. aureus NCTC 8325 reference genome (NC_007795.1) using the RPKM (Reads Per Kilobase transcript per Million reads) method as previously described. Briefly, the number of reads mapping to every ORF are counted. For every ORF, this count is normalized according to the length of the ORF (per kb) and then normalized to the total number of reads in the sample (per million reads). This yields a quantitative absolute measure for the number of mRNAs per gene (the expression level) which is comparable between samples. The RNAseq data was submitted to the European nucleotide Archive ENA; determine the reproducibility between the biological duplicates, scatter plots for each duplicate data sets from each of the three time points were made and the Pearson coefficient was over 0,97 in all cases, indicating good agreement between all duplicates (an example scatter plot is shown in Fig.  and all Pearson coefficients are listed in Table ). To determine how many genes showed altered transcript levels in the presence of ajoene, all fold change values for each time point were sorted by value and plotted in separate charts (Fig. ). Furthermore, the differences between fold change values for all genes were visualized as shown in Fig. . Combined, Fig.  shows that the variation due to technical error and biological variation combined excluded drawing conclusions from fold changes smaller than twofold, and that four-fold changes should be approached with caution. […]

Pipeline specifications

Software tools Primer Express, CLC Assembly Cell, geWorkbench
Databases ENA
Applications RNA-seq analysis, qPCR
Chemicals Sulfur