RNAseq expression analysis of FACS-sorted macrophages from cxcr3.2 mutant and wt zebrafish larvae

The zebrafish Cxcr3.2 is a functional homolog of the human chemokine receptor CXCR3. Zebrafish macrophages lacking this receptor have impaired motility and a rounded shape compared to their wildtype counterparts. To investigate the effects of cxcr3.2 mutation on the transcriptional profile of macrophages, we sorted macrophages from zebrafish larvae lacking a functional cxcr3.2 and compared their transcriptome to that of macrophages from wildtype larvae. Mutant and wildtype macrophages could be clearly distinguished based on the overall differential expression profiles. Classification of genes by compartment showed that peroxisomal, lysosomal and Golgi-related genes were most frequently up-regulated. Moreover, lysosomal and Golgi-related terms were significantly differentially represented in Gene Ontology and KEGG enrichment analysis. Of note, several lysosomal markers (including acidic hydrolases and voltage ATPases) were consistently upregulated in cxcr3.2 mutant macrophages, indicating that cxcr3.2-mediated chemokine signaling is tightly connected to the regulation of lysosomal function. 3 independent groups of 150-200 5dpf Tg (mpeg1: mCherryF cxcr3.2-/- and cxcr3.2+/+) larvae were dissociated and red fluorescent macrophages were FACS sorted. RNA was extracted, retrotranscribed into cDNA and amplified using the SMARTer® Universal Low Input RNA Kit for Sequencing (Clontech), prior Illumina sequencing.