Computational protocol: DNA Phosphorothioate Modification Plays a Role in Peroxides Resistance in Streptomyces lividans

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Protocol publication

[…] Spores of the wild type S. lividans 1326 and the HXY6 were grown in TSBY media to an OD450 reading of 0.3. The cultures were then collected by centrifugation and total RNA was isolated using RNAprotect Bacteria Reagent and RNeasy Mini kits (Qiagen), followed by further purification using a MICROBExpress kit (Ambion) to enrich the mRNA. The resulting mRNA was fragmented using an RNA fragmentation kit (Ambion), producing fragments of sizes ranging from 200 bp to 250 bp. Double-stranded cDNA was generated using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) and purified using a QiaQuick PCR extraction kit. An RNA-seq library was constructed using a Paired End Sample Prep kit (Illumina), and the sequencing was performed using the HiSeq 2000 sequencer (Illumina). Raw reads were filtered to remove dirty reads as recommended by Illumina, and then the resulting clean reads were analyzed with TopHat (version 2.0.5) software (Langmead et al., ), using the annotated S. lividans 1326 genome as reference. We employed R packages edgeR (version3.3; Robinson et al., ) for differential gene expression analysis in this study, using multiple hypothesis test for the p-value correction (q-value), and taking in to account the false discovery rate (FDR). Fold-change was calculated according to the FPKM (Fragments Per Kilobase of exon model per Million mapped reads) value (screening criteria q-value 2 ≤ 0.05, and Fold-change ≥ 2). Gene Ontology (GO) enrichment analysis of differentially expressed genes was analyzed using the GOseq R package, with gene length bias corrected (Young et al., ). GO terms with q-value < 0.05 were considered significantly enriched by differentially expressed genes. […]

Pipeline specifications

Software tools TopHat, edgeR, GOseq
Application RNA-seq analysis
Organisms Streptomyces lividans
Chemicals Diamide, Hydrogen Peroxide, Peroxides, Sulfur