Computational protocol: The Essential Role of H19 Contributing to Cisplatin Resistance by Regulating Glutathione Metabolism in High-Grade Serous Ovarian Cancer

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Protocol publication

[…] All RNA-seq data were aligned to hg19 using TopHat/bowtie with default parameters. The mapped reads were assembled using Cufflinks. Transcript abundances were estimated by Cufflinks in Fragments per Kilobase per Million mapped reads (FPKM) for paired-end reads. Only transcripts with length >200nt were retained. In all differential expression tests, a gene was considered significant if the fold change was more than 2 and q value was less than 0.05. [...] Cells were lysed using lysis buffer (4% SDC in 0.1M Tris-Hcl, ph = 8.0) with SIGMAFAST™ Protease Inhibitor (Sigma, USA). Equal amount of proteins from four cell sublines were reduced with 10 mM DTT and alkylated with 25 mM iodoacetamide. In solution digestion was then carried out with sequencing grade modified trypsin/Lys-C (Promega, USA) at 37 °C overnight. The peptides were acidated with 0.5–1% trifluoroacetic acid (TFA) in the final concentration. The SDC was removed by high speed centrifugation. Tryptic peptides were desalted and centrifuged in a speedvac to dry. Then, Tryptic peptides were redissolved in 0.1% FA.For LC-MS/MS analysis, the peptides were separated by a 90 min gradient elution at a flow rate 0.20 μl/min with a Thermo Scientific EASY-nLC 1000 HPLC system, which was directly interfaced with a Thermo Scientific Q Exactive mass spectrometer. The analytical column was a Thermo Scientific AcclaimR PepMap RSLC column (50 μm ID, 15 cm length, C18, 2 μm, 100 Å). The precolumn was a Thermo Scientific AcclaimR PepMap100 column (100 μm ID, 2 cm length, C18, 5 μm, 100 Å). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of acetonitrile with 0.1% formic acid. The Q Exactive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.2 SP1 software and there was a single full-scan mass spectrum in the orbitrap (350–2000 m/z, 70,000 resolution) followed by 15 data-dependent MS/MS scans at 27% normalized collision energy (HCD). The MS/MS spectra from each LC-MS/MS run were searched against the “human.fasta” from UniProt (release date of March 19, 2014;68406 entries) using Proteome Discoverer software (Version PD1.4, Thermo Scientific, USA). The search criteria were as follows: full tryptic specificity was required; two missed cleavage was allowed; carbamidomethylation (C) were set as the fixed modifications; the oxidation (M) was set as the dynamic modification; precursor mass tolerances were set at 10 ppm; and the fragment mass tolerance was set at 0.02 Da. The peptide false discovery rate (FDR) was calculated using Percolator provided by PD. Relative protein quantification was performed using MaxQuant software (Version 1.4.0.8). Quantitation was carried out only for proteins with 2 or more unique peptide matches and PEP <0.001. Differently expressed proteins were further confirmed by western blotting. […]

Pipeline specifications

Software tools Proteome Discoverer, MaxQuant
Application MS-based untargeted proteomics
Diseases Neoplasms, Ovarian Neoplasms
Chemicals Cisplatin, Glutathione, Hydrogen