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PMD2.G was a gift from Didier Trono (Addgene plasmid # 12259). Plat-A medium was changed 24h post-transfection. The supernatant containing the retroviruses was collected 48h and 72h post-transfection. Debris was eliminated by centrifugation at 2000rpm for 1 minute.
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In/Del realignment and base recalibration were performed using GATK toolbox. Gene counts had been calculated using featureCount , Cufflinks and HTseq . A needle tumor biopsy was extracted at analysis and preserved at -80°C in RNAlater. Mononuclear cells have been isolated by Ficoll-gradient from EDTA-anticoagulated entire blood extracted at diagnosis and preserved at -80°C as dried pellet.
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Prediction of binding to HLA class II molecules of mutated and wild type peptides. Prediction of binding to HLA class I molecules of mutated and wild sort peptides. WES research allowed us to identify a median of 60 mutations (range ) that corresponded to a median TMB of 1.sixty eight Muts/Mb . For those sufferers with materials available for RNAseq we analyzed gene expression and identified these mutated genes with demonstrated expression. This narrowed the number to a median of 35 mutations (range 31-69) .
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