Methods

Ascaroside Treatment: Worms were grown in liquid cultures at 20ºC and 220 RPM using HB101 E. coli as a food source. Worms were synchronously grown in liquid cultures at a density of approximately one worm per uL of liquid and ~75mg/mL HB101 as described in Schaedel et al., (2012). Briefly, for each genotype (N2 and the daf-22 mutant), gravid adults were bleached to obtain a synchronous population of eggs. At six hours post-hatch, the original 60mL culture was separated into six equal 10mL cultures so that all experiments were done in triplicate. The three treatment cultures were each given 150uL of an ascaroside cocktail made of equal amounts (50uL) of 33uM synthetic ascr#2, ascr#3, and ascr#8 in ethanol, for a total ascaroside concentration of 0.5uM in each 10mL liquid culture. The three control cultures were given no ascarosides but were each given 150uL EtOH. The worms in liquid culture were exposed to the ascarosides or solvent for three hours before they were spun down, and the worm pellet was processed using a RNeasy kit to extract and purify the RNA in preparation for sequencing.

Sequencing: RNA integrity was assessed using RNA 6000 Pico Kit for Bioanalyzer and mRNA was isolated using NEBNext Poly(A) mRNA Magnetic Isolation Module. RNA-seq libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacturer’s instructions. Briefly, mRNA isolated from ~1ug of total RNA was fragmented to the average size of 200nt by incubating at 94ºC for 15 minutes in first strand buffer, cDNA was synthesized using random primers and ProtoScript II Reverse Transcripatse followed by second strand synthesis using NEB Second Strand Synthesis Enzyme Mix. Resulting DNA fragments were end-repaired, dA tailed and ligated to NEBNext hairpin adaptors. After ligation, adaptors were converted to the ‘Y’ shape by treating with USER enzyme and DNA fragments were size selected using Agencourt AMPure XP beads to generate fragment sizes between 250 and 300 bp. Adaptor-ligated DNA was PCR amplified followed by AMPure XP bead clean-up. Libraries were quantified with Qubit dsDNA HS Kit and the size distribution was confirmed with High Sensitivity DNA Kit for Bioanalyzer. Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt and sequencing depth of 25 million reads per sample following manufacturer’s instructions.

RNA-seq Data Analysis: Data analysis was performed using the method described in Anders et al., (2013). Briefly, sequence quality control was assessed using ShortRead and a metadata table of the raw fastq data files was created. The reference genome and gene model annotations were obtained from Ensembl (WBcel215), and a reference index was built using bowtie2. Reads were aligned to the reference genome using tophat2 and the BAM files were organized, sorted and indexed before SAM files were created using samtools. Reads were counted using HTSeq-Count and differential expression analysis was performed using edgeR and DESeq. The extended data for both N2 and daf-22 includes the normalized counts for each replicate, the total list of differentially expressed genes, and the subset of significant genes at p = 0.001. Both our raw and processed data can be viewed at the Gene Expression Omnibus (GEO) – Accession Number: GSE171089.