Transcriptomic Analysis of PBL Neo-Epitopes

High quality RNA (1 μg) was subjected to paired-end sequencing using the HiSeq2500 Illumina platform. Complementary DNA libraries were constructed using mRNA-Seq Sample Prep Kit based on the Illumina guide. Library size distribution was validated using Agilent Technologies 2100 Bioanalyzer and quantified by quantitative PCR (KAPA Library Quant Kits, KAPA biosystem). Four normalized sample libraries were pooled together and loaded to a single lane of an Illumina flow cell. Data analysis was performed at the Vital-IT Systems Biology Division, SIB, Lausanne, and at the Lausanne Branch of the Ludwig Institute for Cancer Research. Differential gene expression between groups was performed with DESeq2^{37 (link)} in R. The genes were ranked based on the fold change between patients with or without PBL neo-epitope recognition and this ranked gene list was inputted into GSEA^{38 (link)} to perform gene set enrichment analysis. For this analysis, the enrichment for all the “Canonical pathways” gene sets (version 5.1) from the “Molecular Signature Database”^{39 (link)} was tested (some results are highlighted in Fig. 1i,j and Supplementary Fig. 2; all results with an FDR below 0.05 are reported in Supplementary Table 3).