Gene expression profile of C. elegans biogenic amine synthesis mutants

The goal of this study is to compare the RNA expression profile of wild-type C. elegans nematodes to mutants defective in the synthesis of the biogenic amine neurotransmitters dopamine, serotonin, tyramine, and octopamine in day 2 adults. Overall design: Developmentally synchronized animals were obtained by hypochlorite treatment of gravid adults to release embryos. Synchronized embryos were hatched on NGM plates and grown at 20°C until 48h after the L4 stage of development. Fluorodeoxyuridine was used to prevent the development of second-generation embryos once animals reached fertile adulthood (Gandhi, Santelli et al., 1980). For each RNA-seq experimental replicate, populations were grown simultaneously under the same conditions. Total RNA (typical amount of worms and mass amount of RNA) was isolated from animals using trizol (Invitrogen) combined with Bead Beater lysis in four biological replicates for each genotype, and an mRNA library (single-end, 75-bp reads) was prepared for each sample/replicate using Illumina Truseq with PolyA selection (RUCDR). Libraries were sequenced on an Illumina HiSeq 2500 in Rapid Run Mode, resulting in high quality sequence reads (FAST QC Phred score average of 40 out to the full 75 bp length). Reads (30-40 million per genome and replicate) were mapped tp the C. elegans genome (WS245) and gene counts generated with STAR 2.5.1a. From 93-95% of reads uniquely mapped to the genome. Normalization and statistical analysis on gene counts were performed with EdgeR using generalized linear model functionality and tagwise dispersion estimates. An MDS plot revealed the fourth replicate for each genotype was subject to a strong batch effect, so the fourth replicate samples were dropped, and all analysis re-run using only replicates one, two and three from each genotype. Likelihood ratio tests were conducted for each biogenic amine mutant relative to wild-type odIs77 in a pairwise fashion with a Benjamini and Hochberg correction. Animals carry the odIs77 integrated transgene, which expresses both UbG76V-GFP and mRFP from the col-19 promoter