Young adult (24 h post-vulval crescent L4 stage) whole nematodes were reared at 25°C and exposed to 50 J/m2 UVC radiation then frozen 3h later, experiment carried out in 2007
Young adult (24 h post-vulval crescent L4 stage) nematodes were reared at 25°C and exposed to 50 J/m2 UVC radiation on K-agar plates without food as described (Meyer et al., 2007 Genome Biology 8:R70), placed on OP50-seeded plates for 2 h 45 minutes, washed off plates and rinsed with K-medium 3 times (~5 minutes each) by allowing settling, resuspending, settling again, etc. Nematodes were frozen by dripping 3,000-5,000 pelleted worms suspended in about 500 ul K-medium into liquid nitrogen, and stored at -80°C. The pellets were ground into fine powder with a liquid nitrogen-cooled mortar and pestle and RNA was extracted using an RNeasy kit (Qiagen, Valencia, CA, USA). RNA was quantified with a NanoDrop Fluorospectrometer (NanoDrop Technologies, Wilmington, DE, USA) and analyzed for integrity with a BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
biotin
Label protocol
Starting with 500pg of total RNA, biotin-labeled cRNA was produced using the Epicentre Biotechnologies TargetAmp 2-Round aRNA Amplification Kit 2.0 according to manufacturer's protocol with the exception that 10ul biotin-UTP was incorporated in place of 3.6ul unlabeled UTP, and final product was eluted in 25ul water.
Hybridization protocol
10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol.
Scan protocol
Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using the GenechipÆ Operating Software (Version 1.2.0.037).
Description
UVC radiation exposures were performed in a CL-1000 Ultraviolet Crosslinker (UVP, Upland, CA, USA) with an emission peak at 254 nm. Worms were plated such that they did not shiled each other, and there was no significant K medium remaining at the time of
Data processing
The resulting files (.dat, .cel and .chp) were imported into the Rosetta Resolver system (Version 7.1). This system performs data pre-processing and error modeling as described in Weng (2006). Resolver generated fold-changes and p values, based on ratios built in the system, were exported for further analysis.
