RNA sequencing to identify UNC-49-regulated genes in C. elegans defense against pathogen P. aeruginosa infection

Purpose: To gain molecular insights on how UNC-49 regulates C. elegans innate immunity, we used RNA sequencing to profile gene expression in unc-49(e407) animals relative to wild-type animals with or without P. aeruginosa (PA14) infection. Methods: Synchronized L1 worms were placed onto NGM plates seeded with E. coli OP50 and grown at 20 °C until the L4 stage. Worms were washed off the plates, collected with M9 solution and subsequently transferred to modified NGM plates containing E. coli OP50 or P. aeruginosa PA14 for 24 h at 25 °C. Then, the animals were washed off the plates with M9 solution and frozen in TRIzol (Transgen, ER501-01, China) with liquid nitrogen. Total RNA was isolated using a TransZol Up Plus RNA kit (Transgen, ER501-01, China). Results: RNA seq analyses shows enriched and signficant changed immune genes and pathways that are dependent of GABAergic neuromuscular junctions. Conclusion: Our studies uncover that lack of UNC-49-mediated suppression in unc-49(e407) animals contributes to their improved survival against P. aeruginosa infection. Three replicates of four groups of RNA samples (unc-49(e407) and wild-type L4-stage animals with or without exposure to P. aeruginosa for 24 hrs) were sequenced. Gene expression was compared between groups to identify UNC-49-regulated genes under fed on OP50 or exposed to PA14.