Computational protocol: Electron acceptor redox potential globally regulates transcriptomic profiling in Shewanella decolorationis S12

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Protocol publication

[…] Lactate concentration in culture medium and protein-based biomass volume of the planktonic cells were by HPLC and a protein quantification assay, as previously reported. SigmaPlot 11.0 was used for data (mean and standard deviation) statistical analysis. CLSM was used to analyze the development and structure of the biofilms attached on anode surfaces. Before CLSM observation, biofilm samples were stained with LIVE/DEAD BacLight staining kit (Molecular Probes) which can distinguished viable (with green fluorescence) or unviable or stressed (with red fluorescence) bacterial cells. Randomly sampled view fields were observed and analyzed for each anode biofilm. To obtain three-dimensional structure information, the biofilm sample was observed under the “z-Stack” model of the Zen software (Zeiss). [...] For transcriptome analysis, S. decolorationis S12 cells cultured in LM with different EARPs were collected for RNA extraction as described by Holmes et al.. Planktonic cells were included in transcriptome analysis due to their significant role in electricity generation with electron mediator. RNA protect was immediately added to the cell deposits and then stored at −80 °C before RNA extraction. RNA was extracted using the commercial RNeasy Mini Kit (Qiagen, Germany) with DNase (RNase-free, Takara) treatment according to the manufacturer’s instruction. The extracted RNA was quantified and evaluated by Agilent 2100 bioanalyzer and Caliper Labchip GX. Amplified fragments were sequenced using Illumina HiSeq™ 2500.Raw sequence data were filtered to remove those containing adapter and reads with more than 10% unknown nucleotides, and reads with more than 50% of low quality base (value ≤5). Clean reads were mapped into the transcriptome reference database using Tophat 2.0.1 and Samtools 0.1.18.0. Less than 3 mismatch bases were permitted, and unique mapped reads were obtained. Cufflinks 2.0.0 software was used for calculating the FPKM value and difference analysis. P-value was used to evaluate the difference of gene transcription and FDR was used to determine the threshold of P-value.Genes were annotated in COG (Cluster of Orthologous Groups of protein) database for identification of orthologous proteins and KEGG (Kyoto Encyclopedia of Genes and Genomes) database for annotation and metabolic pathway analysis. Enrichment metabolic pathways were analyzed by both KOBAS 2.0 and Galaxy LEfSe, which set P-value < 0.05 as statistically significant. […]

Pipeline specifications

Software tools SigmaPlot, TopHat, SAMtools, Cufflinks, KOBAS, LEfSe
Databases KEGG
Applications Miscellaneous, Metagenomic sequencing analysis
Organisms Escherichia coli
Chemicals Carbon, Sulfur