ena-STUDY-Klinikum rechts der Isar, II. Medizinische Klinik-30-01-2019-11:06:07:299-80
Bulk 3-prime RNA-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.  Bulk 3'-sequencing of poly(A)-RNA sequencing was performed for 25 diffuse large B-cell lymphoma samples from ITP2-M;Rosa26PB/+;Blmm3/m3 mice. RNA was isolated from whole tissue tumor tissue samples using the Qiagen RNeasy Plus Mini Kit. Library preparation for bulk 3'-sequencing of poly(A)-RNA was done as described in Parekh et al. Scientific Reports 2016. Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher Scientific) using oligo-dT primer containing barcodes, unique molecular identifiers (UMI) and an adapter. 5' ends of the cDNAs were extended by a template switch oligonucleotide (TSO). After pooling of all samples, full-length cDNA was amplified with primers binding to the TSO-site and the adapter. cDNA was tagmented with the Nextera XT kit (Illumina) and 3'-end-fragments finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al. 2016, the P5 and P7 sites were exchanged to allow sequencing for the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 75 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.
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.  Bulk 3'-sequencing of poly(A)-RNA sequencing was performed for 25 diffuse large B-cell lymphoma samples from ITP2-M;Rosa26PB/+;Blmm3/m3 mice. RNA was isolated from whole tissue tumor tissue samples using the Qiagen RNeasy Plus Mini Kit. Library preparation for bulk 3'-sequencing of poly(A)-RNA was done as described in Parekh et al. Scientific Reports 2016. Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher Scientific) using oligo-dT primer containing barcodes, unique molecular identifiers (UMI) and an adapter. 5' ends of the cDNAs were extended by a template switch oligonucleotide (TSO). After pooling of all samples, full-length cDNA was amplified with primers binding to the TSO-site and the adapter. cDNA was tagmented with the Nextera XT kit (Illumina) and 3'-end-fragments finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al. 2016, the P5 and P7 sites were exchanged to allow sequencing for the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 75 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.