B-cell receptor repertoire sequencing of murine diffuse large B-cell lymphoma samples (PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice)

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. In this study, we describe novel PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate newly discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive novel insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology. Analysis of the B-cell receptor repertoire was performed for 30 diffuse large B-cell lymphoma samples from ITP2-M;Rosa26PB/+;Blmm3/m3 mice. Library preparation was conducted as described in Turchaninova et al. Nature Protocols 2016 with minor modifications, which allowed us to introduce Illumina Nextera adapters and indexes during PCR. Briefly, 700 ng RNA (extracted from whole tissue lysate) was transcribed into cDNA using a 5' template switch oligonucleotide, which contains a unique molecular identifier. Immunoglobulin heavy (IGH) and light chain (IGL) libraries were amplified using a set of IGHC-specific / IGLC-specific and 5' template switch oligonucleotide-specific primers introducing indexed Nextera sequencing adapters. The resulting libraries were analyzed on the Illumina MiSeq (300 bp paired end).