Computational protocol: Integrative “Omics”-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses

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Protocol publication

[…] Heavy labeled and non-labeled cells were completely lysed in SDS-Buffer (4% SDS in 100 mM Tris/HCl pH 7.6) and shortly heated at 95°C. Sonication was performed for DNA sharing prior to sample centrifugation at 16,000g for 5 min. Protein concentration of the clear supernatant was measured by DC protein assay (Biorad) to mix labeled and non-labeled protein samples in the same amount. To reduce sample complexity for MS-analysis, proteins were separated by SDS-PAGE (NuPAGE 4%–12% Bis-Tris gel, Invitrogen) and stained with Colloidal Blue Staining Kit (Invitrogen). Each lane was cut into 14 gel pieces for in-gel digestion as described . In brief, proteins were reduced by 50 mM dithiothreitol (DTT), alkylated with 550 mM iodoacetamide and digested with the endopeptidase Lys-C (enzyme to protein ratio 1∶100, Wako). After digestion and elution, peptides were desalted by stop and go extraction (STAGE) tips . Next to in-gel digestion, samples were also digested in solution as described .For LC-MS/MS, a nano liquid chromatography (LC) system (Thermo Fisher Scientific) was coupled to a LTQ-Orbitrap Velos or a Q-Exactive mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Proxeon). Fused silica emitter were packed in-house with C18-AQ RepoSil-Pur (3 µm, Dr. Maisch GmbH) and used as columns for reverse-phase chromatography to separate peptides by a linear gradient of 5–30% acetonitril with 0.5% acetic acid for 150 or 240 min at a flow rate of 200 nl min−1. After elution, peptides were ionized and transferred to gas-phase by electrospray ionization (ESI) to enter the mass spectrometer. For measurements with the LTQ-Orbitrap Velos mass spectrometer, full MS scan spectra (m/z = 300–1650) were acquired in the Orbitrap with a resolution of R = 60,000 after accumulation of 1,000,000 ions. The 15 most intense peaks from full MS scan were isolated and fragmented in the linear ion trap after accumulation of 5,000 ions. Fragmentation of precursor ions was performed using CID (35% normalized collision energy) prior to acquisition of MS/MS scan spectra. Q-Exactive measurements were performed as described . The 10 most intense peaks were selected and fragmented by higher energy collisional dissociation.Analysis of raw data was performed by the MaxQuant software package (version 1.2.2.9) as described . Database searches were performed with the Andromeda search engine against a house-made R. sphaeroides 2.4.1 database. The two chromosomes and five plasmids of R. sphaeroides 2.4.1 were translated into protein sequences using EMBOSS Transeq . Translation was performed for all six reading frames. A unique identifier indicating the sequence position and frame was generated and assigned to each resulting open reading frame (ORF) longer than six amino acids. The generated ORF database was combined with all public available protein sequences for R. sphaeroides 2.4.1 and then used for the peptide identification step. By applying a decoy approach we determined the false discovery rate (FDR) to be smaller than 1%. After peptide identification, database entries belonging either to the de novo generated ORF set or to the public available annotated proteins were clustered into protein groups (MaxQuant). Groups lacking an annotated member were assumed to be potentially new coding sequences and were selected for further investigation.Detection and quantification of SILAC pairs was performed by MaxQuant using following parameters: Lys-C as digesting enzyme with a maximum of two missed cleavages, carbamidomethylation of cysteins as fixed modification, oxidation of methionine and acetylation of the protein N-terminus as variable modifications, SILAC amino acid labeling: Lys6. Maximum mass deviation was set to 7 ppm for the peptide mass and 0.5 Da for MS/MS ions. For identification of peptides and proteins a FDR of 1% were used and only peptides with minimum of six amino acids length were considered for identification. For SILAC analysis, two ratio counts were set as a minimum for quantification. Bioinformatic analysis was performed with Perseus (version 1.3.0.4) to calculate p-values with a Benjamini-Hochberg multiple testing correction based on a FDR threshold of 0.05. [...] For gene expression studies, isolated RNA was hybridized to Custom Gene Expression Microarrays from Agilent Technologies (8x15K; ID: 027061) designed for R. sphaeroides wild-type 2.4.1 . The arrays contain oligodeoxynucleotide probes (60-mers) for 4304 open reading frames, according to genome annotations available on the IMG server (Integrated Microbial Genomes; img.jgi.doe.gov/cgi-bin/w/main.cgi), and for 144 putative sRNAs identified in our group , . Two µg RNA from reference (no stress) and stress samples were chemically labeled with Cy5 and Cy3, respectively, using the ULS Fluorescent Labeling Kit for Agilent arrays (Kreatech) and competitively hybridized to arrays (two-color microarrays). Fragmentation of labeled RNA, hybridization to arrays, and washing was performed using the Gene Expression Hybridization and Wash Buffer Kits according to the specifications of Agilent. Hybridization was performed at 65°C for 17 hours. Read-out files for arrays were generated with the Agilent DNA microarray scanner, followed by compilation of raw median fluorescence values using the Feature Extraction Software (Agilent). Within-array normalization according to LOESS was accomplished with the Bioconductor package Limma for R . Those values were retained that exhibited an average signal intensity (A-value: 1/2 log2 (Cy3×Cy5)) above background, as specified by Agilent control probes present on each array (Poly 90 min A≥10.44; Total 7 min A≥10.27; Total 45 min A≥10.61, Total 90 min A≥10.45). Fold changes were calculated from remaining values as log2 ratios (Cy3/Cy5). Data shown in this study represent the results from two individual microarrays (biological replicates), each containing a pool of three independent experiments for each sample. Statistical analysis was performed by Perl Statistics modules. Targets having p-values <0.05 and log2 ratios ≥0.8 or ≤−0.8 were assumed to represent deregulated candidates. For expression cluster analysis, log2 ratios were imported to MeV (Multi Experiment Viewer version 4.7.4) from the TM4 Microarray Software Suite , and visualized as heat-maps. Clustering was based on k-means (KMC method) according to Euclidean distance with a maximum of 50 iterations.The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE42244 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE42244). [...] RNA for sequencing was isolated as described for microarray analysis. R. sphaeroides 2.4.1 cultures were grown under semi-aerobic conditions to a final OD660 of 0.4. The cDNA libraries were prepared at Vertis Biotechnology AG (Germany). For this, the RNA samples were poly(A)-tailed by poly(A) polymerase. After that, the 5′-PPP residues were removed using tobacco acid pyrophosphatase (TAP) followed by the ligation of the RNA adapter to the 5′-phosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 20–30 ng µl−1 using a high fidelity DNA polymerase. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The following adapters sequences flank the cDNA inserts (the NNNNNN indicates the barcode sequence used for multiplexing): 5′-end: 5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T-3′ and 3′-end: 5′-CAA GCA GAA GAC GGC ATA CGA GAT-NNN NNN-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TTT TTT TTT TTT TTT TTT TTT TTT T-3′. The cDNA libraries were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics), analyzed by capillary electrophoresis and finally sequenced by an Illumina GAIIx machine.The sequences of the obtained sequencing reads were quality trimmed by the program fastq_quality_trimmer from the FASTX program suite with a cut-off phred score of 20. Poly(A) tail sequences were clipped from the 3′ end of the sequences, the resulting sequences were filtered by length and sequences short than 12 nt were discarded. The remaining reads were aligned to the reference genome sequences (accession numbers: CP000143.1, CP000144.1, CP000145.1, CP000146.1, CP000147.1, DQ232586.1, DQ232587.1) using the short read mapper segemehl . Based on these read mapping, coverage plots which represent the number of mapped reads per nucleotide were created. Those were visualized and examined in the Integrated Genome Browser . […]

Pipeline specifications

Software tools Segemehl, IGB
Application RNA-seq analysis
Organisms Rhodobacter sphaeroides
Chemicals Oxygen