Sample is also known as m577 5 Genotype is glp-4(bn2); daf-2(m577) Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria. Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com). All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre. Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org). Keywords = Caenorhabditis, elegans, aging
