Bulk RNA-sequencing on sorted zebrafish macrophages based on novel irg1-knock-in reporter comparing control versus nlrc3l mutation under baseline and injury conditions
Using a GFP knock-in zebrafish targeting the irg1/acod1 locus, a gene highly induced in activated macrophages, we isolated macrophages based on high and low GFP levels by FACS to conduct bulk RNA-sequencing. Transcriptome analysis of macrophages showed nlrc3l mutants had constitutively activated macrophages that sustained elevated expression of pro-inflammatory genes, including irg1 and tnfa, and canonical and non-canonical NF-?B pathways genes. Furthermore, transcriptome analysis suggested that wild-type/control macrophages exhibited hybrid M1-like and M2-like characteristics during initial injury response, while nlrc3l mutant macrophages remained locked in a pro-inflammatory state. This analysis supports the rest of the functional experiments to demonstrate that chronic macrophage activation restricts functional plasticity associated with mrc1b deficiency and thereby hinders skeletal muscle repair. Overall design: Two independent RNA-seq datasets were generated in this study. One dataset (Samples 1-12) corresponds to RNA-seq profiling of macrophages sorted from 3dpf zebrafish embryos that were of either sibling or mutant nlrc3l/st73 genotype based on low or high GFP levels indicating endogenous irg1 transcriptional levels using a GFP knock-in reporter at the irg1 locus. Second dataset (Samples 13-19) corresponds to RNA-seq profiling of macrophages that were similarly isolated based on relative expression of the GFP knock-in reporter for endogenous irg1 from injured (24-hour-post major tail amputation) and non-injured 3dpf zebrafish embryos from control and nlrc3l mutant genotypes.