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Pipeline publication

[…] 5 min at 4°C. The pellet was reextracted by the use of 1 ml cold sterile water. Supernatants from the two extractions were combined and stored at −80°C for NMR analysis., The collected supernatants were lyophilized and reconstructed in NMR buffer (KH2PO4/K2HPO4 buffer–D2O [pH 7.4] [uncorrected], with 500 μM trimethylsilylpropanoic acid [TMSP] as an internal standard). Two-dimensional (2D) 1H-13C heteronuclear single-quantum coherence (HSQC) spectra were collected on a Bruker DRX Avance 500-MHz spectrometer equipped with a 5-mm-long triple-resonance cryoprobe (1H, 13C, and 15N) with a z axis gradient, a BACS-120 sample changer, Bruker IconNMR, and an automatic tuning and matching (ATM) unit. NMRPipe () and NMRViewJ () were used to process and analyze the collected spectra. The TMSP internal standard was used for chemical shift referencing and normalization of NMR peak intensities. NMR peaks from the 2D 1H-13C HSQC spectra were annotated by comparing the observed 1H and 13C chemical shifts to the metabolite reference data from the Platform for RIKEN metabolomics (PRIMe; http://prime.psc.riken.jp/) (), Human Metabolome Database (HMDB; http://www.hmdb.ca/) (), Madison metabolomics Consortium Database (http://mmcd.nmrfam.wisc.edu/) (), Metabominer (http://wishart.biology.ualberta.ca/metabominer/) (), and BiomagResBank (BMRB; http://www.bmrb.wisc.edu//) () with error tolerances of 0.08 ppm and 0.25 ppm for 1H and 13C chemical shifts, respectively. The relative intensity (i.e., concentration) of each metabolite was calculated by averaging the intensities of all NMR peaks unambiguously assigned to the metabolite., Cultures of S. aureus JE2 and JE2 ccpA strains were grown overnight in CDM and CDMG and inoculated to an OD600 of 0.05 into TSB (TSB overnight), CDM, and CDMG, respectively. Cultures were grown at 37°C in a 1:10 vol-to-flask ratio at 250 rpm. Fifteen milliliters of bacterial culture was collected at the mid-exponent […]

Pipeline specifications

Software tools NMRPipe, NMRViewJ, MetaboMiner