Computational protocol: Phenotypic responses to interspecies competition and commensalism in a naturally-derived microbial co-culture

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Protocol publication

[…] Cells were collected by  centrifugation (7000 RPM, 4 min at 4 °C) from 50–100 ml of cell broth sampled from the mid-log phase (20 and 33 h) of each axenic and co-culture treatment (plus or minus glucose or xylose, respectively). The cell pellets were immediately flash frozen in liquid nitrogen and stored at −80 °C to await RNA isolation. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA), followed by genomic DNA removal and cleaning using RNase-Free DNase Set kit and the Mini RNeasy kit (Qiagen, Hilden, Germany). Ribosomal RNA was depleted from each sample using Ribo-Zero (bacteria) rRNA Removal Kit (Illumina, San Diego, CA). The quality and integrity of the RNA was assessed on an Agilent 2100 Bioanalyzer; only samples with integrity numbers between 8 and 10 were selected for further analysis. Template cDNA was prepared using the Applied Biosystems SOLiD Total RNA-Seq Kit (Life Technologies, Carlsbad, CA) according to manufacturer’s protocol. Sequencing was carried out using the SOLiD 5500XL protocol (Life Technologies Carlsbad, CA). The 50-base sequence reads were mapped to the genomes of Halomonas HL-48 and Marinobacter HL-58 using SOLiD LifeScope v. 2.5 software. The complete reference genomes have previously been published. Nucleotide sequence is available through the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under accessions GCA_000686925.1 (Halomonas HL-48) and GCA_000686085.1 (Marinobacter HL-58). The genomes were annotated integrating information from the following pipelines: 1) the DOE-JGI Microbial Genome Annotation Pipeline, BlastKOALA, and the RAST server, as previously described. Annotations are available on GitHub (see below) and through IMG (http://img.jgi.doe.gov). The total number of protein encoding genes identified for Halomonas HL-48 and Marinobacter HL-58 were 3381 and 3875, respectively. RNA-seq was performed on 3–5 biological replicates. After alignment raw counts were normalized and differentially expressed genes were identified using the R package DESeq2. Differentially expressed genes are defined as those showing > 2-fold change in expression when comparing two conditions with an adjusted p-value of ≤0.05. […]

Pipeline specifications

Software tools LifeScope, BlastKOALA, RAST, DESeq2
Databases ENA
Application RNA-seq analysis
Organisms Escherichia coli, Bacteria
Chemicals Carbon, Glucose