Computational protocol: The Use of CRISPR/Cas9 Gene Editing to Confirm Congenic Contaminations in Host-Pathogen Interaction Studies

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[…] 2.5 × 106 BMMs were infected with S. Typhimurium as described in the gentamicin assay section (MOI 5). At 6 h post-infection, cells were washed in cold PBS before being harvested using a cell scraper in cold PBS. Cells were spun down, and pellets were snap-frozen in liquid nitrogen before being stored at −80°C prior to subsequent analysis. Cell pellets were lysed and processed by filter-aided sample preparation (FASP), as previously described (Wisniewski et al., ). Briefly, cells were lysed in 4% SDS, 0.1M Tris-HCl (pH 7.6), sonicated and then reduced with 5 mM tris(2-carboxyethyl)phosphine (TCEP) at 60°C for 30 min before being passed over a FASP column (Expedeon). Samples were alkylated and subjected to tryptic digestion on-column overnight at 37°C according to the manufacturer's instructions. Prior to mass spectrometry, FASP eluates were acidified and peptides were further purified and enriched using OMIX C18 Mini-Bed tips (Agilent Technologies). Using a Dionex UltiMate 3000 RSLCnano system equipped with a Dionex UltiMate 3000 RS autosampler, an Acclaim PepMap RSLC analytical column (75 μm × 50 cm, nanoViper, C18, 2 μm, 100Å; Thermo Scientific) and an Acclaim PepMap 100 trap column (100 μm × 2 cm, nanoViper, C18, 5 μm, 100Å; Thermo Scientific), tryptic peptides were separated by increasing concentrations of 80% ACN/0.1% formic acid at a flow of 250 nL/min for 95 min and analyzed with a QExactive Plus mass spectrometer (Thermo Scientific). Data was searched against the Mus musculus reference proteome (revision as of 2016-08), canonical and isoform sequences (consisting of 59,550 entries) using Maxquant (v1.5.2.8) (Cox and Mann, ; Cox et al., ) with the following settings: enzyme digestion set to trypsin (max missed cleavages of two); fixed carbamidomethyl cysteine modification; max variable modifications of five; minimum peptide length of 8 amino acids, maximum peptide length of 25 amino acids; known contaminants added to the search database; decoy database set to reverse; 1% false discovery rate (FDR) for peptide and protein identification; instrument parameters (including ppm tolerance settings) were the default settings for Orbitrap acquisition; label free quantitation (LFQ) was set to on and with a minimum ratio count of 2. For LFQ analysis, the Perseus (v1.5.3) software package was used (Tyanova et al., ). Raw LFQ values were Log2-transformed, reverse and contaminant sequences removed and rows set to require a minimum of three valid values across the six samples (three wild-type and three Tlr7−/−) (Supplementary Table ). Any further missing values were replaced by imputation according to the normal distribution of the samples, with default values of width (0.3) and down shift (1.8) selected (Supplementary Table ). Data were also analyzed by z-score normalization across each sample row (Supplementary Table ). For a list of all identified peptides and assigned protein groups and MS metadata, refer to Supplementary Tables , . Raw MS data has been deposited on the ProteomeXchange PRIDE (PRoteomics IDEntifications database at the European Bioinformatics Institute) (Vizcaino et al., ) archive and can be accessed using the following accession number: PXD007239. […]

Pipeline specifications

Software tools MaxQuant, Perseus
Databases PRIDE ProteomeXchange
Application MS-based untargeted proteomics
Organisms Mus musculus, Salmonella enterica subsp. enterica serovar Typhimurium, Bacteria
Diseases Bacterial Infections, Salmonella Infections