MYD88 L265P differential expression analysis

The wider transcriptional effects of MYD88L265P were explored by analysing the microarray datasets using the limma package. We focussed on evidence for differential expression between Myd88L265P and Card11L232LI transduced B cells because both cell populations were actively proliferating at the time of RNA isolation. When the transcriptome of MYD88L265P expressing B cells was compared with empty vector-expressing B cells using limma, 111 genes had strong evidence for differential expression with 88 increased in MYD88L265P cells. Compared to CARD11L232LI expressing B cells, 84 genes had strong evidence for differential expression in MYD88L265P B cells with 30 increased, including the plasma cell induced genes Prdm1 and Igj. Duplicates of transduced B cells, including empty vector controls were cultured without anti-CD40 for 24hr and EGFP+ cells sorted using FACSAria cell sorter. For microarray analysis, sorted cells were resuspended in TRIzol and RNA extracted. mRNA expression was analyzed on Affymetrix mouse ST 1.0 arrays as per the manufacturer’s instructions. Microarray data were subjected to quality control, normalization and differential expression analysis in limma.