Using single-worm RNA sequencing to study C. elegans responses to pathogen infection

We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and applied this method to study C. elegans responses to pathogen infection. Five replicates of two groups of samples (five worms with or without exposure to P. aeruginosa for 24 hours) were collected and subjected to mechanical worm lysis, RNA isolation, cDNA synthesis, library preparation, sequencing, and sequence data analysis. At each step, the results of the single-worm RNA-seq were compared to those of standard RNA-seq. The sequence data (FASTQ files) of standard RNA-seq were generated in the study of Sellegounder et al. (Science Advances 2019; 5(11):eaaw4717) and were retrieved from the NCBI’s SRA database through the GEO using the accession number GSE122544. These sequence data (from N2_OP50 and N2_PA14 samples) were re-analyzed in the current study in the same way as in the single-worm RNA-seq.