RNA sequencing

Synchronized WT, alg-1(gk214) and alg-2(ok304) animals cultured at 20°C were collected at adult day 5 after removing eggs and progeny larvae. Adult C. elegans were separated from eggs and progeny on a daily basis by washing plates with M9 into conical tubes and allowing the adults to settle by gravity for a few minutes on a bench top. The supernatant containing larvae and eggs was then removed, and this process was repeated 3–7 times until eggs and larvae were no longer visible. Three independent RNA samples of each strain were prepared for RNA sequencing with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) according to the Low Sample Protocol. 50-bp single-end indexed RNA sequencing libraries were prepared from 1 μg of RNA of each sample and used for sequencing on an Illumina HiSeq platform. Subsequent mapping of sequencing reads to the C. elegans genome (ce10) was performed using RNA-STAR [66]. Total read counts for each gene were then quantified using HTSeq [67]. These read counts were then input into DESeq [68] to determine log2-fold change and differential expression between the mutant and WT strains.