Computational protocol: A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus

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Protocol publication

[…] Delipidized protein samples with disulfide bonds reduced and thiol groups alkylated were trypsinized and further processed for nano-LC-MS/MS using the Q Exactive Orbitrap mass spectrometer (Thermo Fisher). Raw data were analyzed using MaxQuant, with protein localization and function predicted using LocateP and DAVID. [...] Following MV isolation from early-stationary-phase GAS cultures, samples were treated with Benzonase nuclease (Sigma) for 30 min to degrade all forms of extracellular nucleic acids prior to being washed once with PBS. Vesicular RNA was isolated from biological triplicates using TRI reagent (Sigma), followed by treatment with DNA-free Turbo DNase (Ambion) to remove any contaminating genomic DNA (gDNA). The RNA concentration was assessed using a Bioanalyzer (Agilent) prior to library construction. cDNA libraries were generated without rRNA depletion using the ScriptSeq v2 library preparation kit (Epicentre) and sequenced on an Illumina HiSeq 2500 platform. Short or low-quality reads were filtered and trimmed prior to mapping of trimmed reads to the most closely related reference genome (MGAS5005; GenBank accession no. NC_007297) using the STAR alignment tool (). Visualization and manual inspection of read coverage were conducted using the Integrative Genomics Viewer (IGV) (). Differentially abundant RNAs in MV samples and bacteria were computed using the DESeq2 package (). Prophage-associated genes (φ5005.1, M5005_0995 to -M5005_1054, φ5005.2, M5005_1168 to M5005_1222; and φ5005.3, M5005_1414 to -M5005_1467) were removed from differential expression results. […]

Pipeline specifications

Software tools STAR, IGV, DESeq2
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Fasciitis, Drug-Related Side Effects and Adverse Reactions
Chemicals Cysteine