Affymetrix Generic Hybridization
The Affymetrix raw expression CEL files were processed using R and Bioconductor (R Development Core team, 2011; Gentleman, 2004). All files were first normalized together using RMA (Bolstad et al., 2003), and control probes were excluded for further analysis.
Total RNA was sent to the GeneCore facilty in Heidelberg, Germany (http://genecore3.genecore.embl.de/genecore3/index.cfm), and processed according to their standard protocols.
Animals were placed at the restrictive temperature (25C) for 0, 6, 12 or 24 hours.
Synchronised cultures of pash-1(mj100) and pash-1(mj100); pash-1::gfp animals were grown to the late L4 stage at 15C. At this time a fraction of the animals in each sample were harvested, and the remainder placed at 25C, to be harvested at 6, 12 and 24 hours subsequently. This experiment was repeated three times for each genotype.
For total RNA isolation animals were harvested from plates by washing with M9. Animals were pelleted and frozen in liquid nitrogen and dissolved in ten pellet volumes of Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted according to the manufacturer's protocol.