Comprehensive transcriptome characterization deciphers mechanisms underpinning the positive selection of B cells in germinal centers. [mouse; short-read]

Alternative splicing (AS) and translational regulation play major roles in the activation and differentiation of immune cells. Previous studies using short-read sequencing linked individual AS events in germinal center (GC) B cells to proliferation and survival. To establish a more comprehensive understanding of transcript isoform changes that accompany B cell selection in the GC we developed a workflow for long-read nanopore sequencing. MYC expression by GC B cells marks cells undergoing a programme of AS which includes the removal of poison exons (PE) in splicing factors SRSF3 and SRSF7. These are crucial for B cell expansion and regulate transcripts encoding the translation machinery which is markedly increased upon positive selection of the GC B cell. Overall design: Short-read (Illumina) RNA-seq data from naïve and subsets of germinal centre B cells, from mouse. A total of 5 replicates were included for each cell type; however, note that mouse LZ GFP-cMYCneg rep3, and LZ GFP-cMYCpos rep 1 and rep 2 library preparation initially failed, and the repeated libraries were found to be low quality. These samples were therefore excluded from downstream analysis for the short-read data, leaving a total of 3-5 replicates for each cell type.