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Sequencing of Caenorhabditis elegans wildtype strain (N2) treated with T25B9.1 RNAi for 5 days after L4 larvae stage.

ABSTRACT: Sequencing of Caenorhabditis elegans wildtype strain (N2) treated with T25B9.1 RNAi for 5 days after L4 larvae stage.

PROVIDER: PRJNA431728 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6513874.fastq.gz	Fastqsanger.gz
	SRR6513875.fastq.gz	Fastqsanger.gz
	SRR6513876.fastq.gz	Fastqsanger.gz
	SRR6513877.fastq.gz	Fastqsanger.gz
	SRR6513878.fastq.gz	Fastqsanger.gz

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Project description:Acyl-coA synthases (ACSs) produce fatty acyl-CoAs that are used in metabolic and signaling pathways. Metazoans have a large number of ACS genes with differing expression patterns and substrate preferences, but the physiological roles of most ACS genes are unknown. Here, we focused on the C. elegans acyl CoA synthase, ACS-3, which is known to regulate fat uptake and de novo fat synthesis through the conserved nuclear hormone receptor, nhr-25. We performed microarray analysis of acs-3 mutants to elucidate the acs-3-regulated transcription program. This analysis revealed an enrichment among differentially regulated genes of those involved in lipid metabolism, pathogen and wounding responses, and sterol binding genes, among others. As the immunity genes were the most represented gene class, we performed pathogen sensitivity assays to test the phenotypic consequences of this immune gene regulation. Interestingly, acs-3 mutants were hypersensitive to the fungal pathogen D. coniospora, but only mildly sensitive to the bacterial pathogen P. aeruginosa. acs-3 mutation suppressed nhr-25 mutant sensitivity to P. aeruginosa, yet surprisingly microarray analysis of nhr-25(RNAi) animals revealed significant overlap with the acs-3 mutant transcriptome, with an enrichment of pathogen response genes. The upregulation of pathogen response genes in acs-3(ft5) mutants and following nhr-25 reduction-of-function (rf) does not appear to be due to a constitutive osmotic response or defective cuticle barrier, two potential explanations for the acs-3(ft5) and nhr-25(rf) expression of innate immunity genes in the absence of pathogen exposure. Together, these data indicate that ACS-3 promotes resistance to the fungal pathogen, D. coniospora and regulates innate immunity genes through an unknown mechanism. Potential roles for ACS-3 in innate immunity are discussed. We used two-color expression microarrays to compare the transcriptional profiles in two experimental conditions: 1) comparing wild-type (N2) L4 stage larval worms to acs-3(ft5) L4 larval mutant animals; and 2) animals grown to L4 larval stage on bacteria harboring vectors for either control or nhr-25 RNA-interference (RNAi). L4 stage was determined by morphology of the developing vulva. Three biological replicates were used for each experimental condition. Statistically significant changes in gene expression in each experimental were determined using M-bM-^@M-^\linear models for microarray dataM-bM-^@M-^] (limma).

Project description:Purpose: The objective of this study was to identify genes whose expression changes significantly between the L4 larval stage (Day 0 of adulthood) and the sixth day of adulthood (Day 6) in C. elegans. Methods: Wild type N2 animals were synchronized by sodium hypochlorite treatment and dropped as L1 larvae to NGM RNAi plates containing 2mM IPTG, 100 mg/mL carbenicillin and seeded with E. coli strain HT115 harboring the L4440 plasmid (vector only control for feeding-based RNAi experiments). Animals were maintained on these plates at 20˚C until the L4 larval stage when half of the cohort was harvested for total RNA (brief description to follow). The other half of the cohort was transferred to new NGM RNAi plates containing 2mM IPTG, 100 mg/mL carbenicillin, 25ug/mL FUDR and seeded with E. coli strain HT115 harboring the L4440 plasmid. C. elegans were maintained on these plates at 20˚C until they reached the sixth day of adulthood when they were harvested for total RNA. To isolate RNA, worms were washed off of plates in M9 buffer, pelletted by centrifugation and resuspended in Trizol. Following a phenol/chloroform extraction, total RNA was precipitated in isopropanol, washed in 70% ethanol, and resuspended in RNase-free H2O. Total RNA was treated with DNase and reisolated using Qiagen RNeasy columns. RNA was isolated from two independent cohorts of L4 animals and three independent cohorts of Day 6 adults. rRNA was depleted from total RNA using Ribo-Zero and libraries were prepared by strand-specific cDNA synthesis for Illumina HiSeq sequencing. Following quality control steps to check for ribosomal depletion, contamination, and leftover adapter sequences, sequences were trimmed and alinged to the C. elegans genome using TopHat2. Alignments were then run with featureCounts then normalized using edgeR's calcNormFactors then transformmed using limma's voom. Changes in gene expression were identified by performing a differential expression analysis using DESeq2 on the Igenbio ERGO platform (Igenbio, Inc., Chicago, Il). Results: Results from DESeq2 were filtered for statistically significant changes in expression by accepting only those changes with a q-value of less than 0.05. As compared to expression at the L4 larval stage, at Day 6 of adulthood we found there to be 1441 genes that were upregulated by more than 5-fold and 2660 genes were downregulated by more than 5-fold. Conclusions: Aging is commensurate with significant and substantial changes in gene expression in C. elegans. By the time adults enter the post-reproductive phase of their life, we found that approximately 4000 genes are differentially expressed as compared to their expression levels in L4 larvae. While some of these changes in expression may drive the aging process, others may protect animals from age-associated damage.

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Sequencing of Caenorhabditis elegans wildtype strain (N2) treated with T25B9.1 RNAi for 5 days after L4 larvae stage.

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