Polysome profiling and mRNA-seq to quantify translational gene regulation in dietary restricted C. elegans and in a long-lived daf-2:rsks-1 double mutant.

Dietary restriction (DR) and loss of the genes rsks-1 and daf-2 increase longevity in C. elegans. Polysome profiling allows actively translated mRNA bound by multiple ribosomes to be isolated and compared to the total mRNA present. In this project, we differentiate transcriptional and translational changes in gene expression in C. elegans by combining polysome profiling and mRNA-sequencing. By comparing gene abundance between the two RNA pools, genes with altered translational regulation under DR and in the mutant can be identified. Overall design: Total and polysome bound RNA was isolated from well-fed daf-2;rsks-1 double mutants, well-fed N2, and dietary restricted N2 C. elegans in biological quadruplicate. Age syncronized worms were maintained at 20°C and subject to DR starting at day one of adulthood in the presence of FuDR to prevent contamination of progeny. Whole worms were lysed at day four of adulhood and the lysate was subject to polysome profiling to isolate RNA bound to 2 or more ribosomes. A aliquot of unprocessed lysate was used for RNA extraction of total RNA. RNA was enriched for polyadenylated transcripts and used for unstranded paired-end library synthesis with a Tru-seq RNA kit. Read length of 100 bp were generated using Illumina HiSeq 2000. Reads were aligned to the C.elegans genome as guided by gene annotations from ensemble version 66. Processed data contains the unfiltered counts per million (CPM) of normalized reads aligning to each gene for each of the samples submitted.

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Lan J, Rollins JA, Zang X, Wu D, Zou L, Wang Z, Ye C, Wu Z, Kapahi P, Rogers AN, Chen D