Before each of the experiments, we cultured the worms for at least three generations at 20°C without allowing the worms to deplete their food. Other than the 25°C treatments, done in a 25±0.5° incubator, all incubations and procedures involving live worms were done in a temperature controlled room at 20±0.3°C. To minimize temperature variation during handling, we used large (10 cm diameter) plates, wore insulated gloves while handling plates, and used an external light source for the dissecting microscope. For the microarray experiment, we used a platinum wire loop to transfer several hundred embryos to fresh plates for each generation. For the multigenerational follow-up experiments (not included in this accession because they were analyzed by RT-QPCR only), we used a platinum wire pick to transfer 60 L4 larvae to fresh plates for each generation. The purpose of transferring L4s instead of embryos in the multigenerational experiment was to eliminate males from the cultures, thereby eliminating variation in the frequency of sex-specific imprinting (most C. elegans individuals are hermaphrodites, with a low rate of spontaneous males that increases with temperature). For the same set of experiments, we used HOBO miniature temperature loggers (Onset) to continuously monitor culture temperatures. For post-25° steps of all experiments, we interleaved the constant 20° (control) plates with post-25° treatment plates in common stacks whenever possible, so that treatment and control plates experience the same microenvironment on average. In practice, the treatment plates were slightly staggered in time relative to the corresponding control plates of the same generation, because of time constraints and because development time from zygote to adult is ~17 hours faster at 25° than at 20°. For the each condition and generation in the microarray experiment, we collected 3 replicates (50 embryos each replicate) of the standard C. elegans laboratory strain N2 and three replicates (50 embryos each replicate) of the strain HC445 (sid-1(qt9) in an N2 background).
Growth protocol
We used standard methods to culture and handle C. elegans (Brenner, 1974), except we used the sporulation-defective Bacillus subtilis strain RL1275 (spoIIAC::erm in a PY79 background, kind gift from Richard Losick) as worm food, because B. subtilis is substantially less pathogenic to C. elegans than the standard C. elegans food E. coli OP50 at 25°C (Garsin et al., 2003).
Extracted molecule
polyA RNA
Extraction protocol
We collected embryos and purified RNA as described (Baugh et al., 2003), except we increased the number of embryos per sample to 50. In brief, we picked young adults into water, chopped them in half, treated them with alkaline bleach to destroy any RNA and protein not protected by the eggshell, stopped the bleach reaction with bovine serum albumin, collected and washed early embryos in DEPC-treated water using a glass mouth pipette, flash-froze the embryos in liquid nitrogen in low-adhesion 600 μl microcentrifuge tubes, and transferred the tubes to a -80° freezer for storage. For the microarray experiment, the total time from picking of the first worm to bleaching was not allowed to exceed 15 min. For all subsequent experiments, the time was not allowed to exceed 8 min. We added Trizol (phenol/guanidine isothiocyanate, Invitrogen), with added linear polyacrylamide and yeast tRNA as carriers, to the frozen embryos for RNA extraction and isopropanol precipitation. We used the RNA pellet directly for RT (reverse transcription), starting from a T7-oligo-dT RT primer. To obtain sufficient RNA for microarray analysis, RNA was amplified by two rounds of in vitro transcription (IVT) using the MessageAmpII kit (Ambion) with the following modifications designed to increase the average lengths of the aRNA populations (Baugh et al. 2001): First-round cDNA construction from total RNA was carried out at 1/5 of the recommended quantities and total volume, while IVT was carried out at 1/2 of the recommended volume. 100ng aRNA from the first round was used as input for the second round of amplification following the manufacturers protocol for amino-allyl modified nucleotides.
Label
cy3
Label protocol
10 µg aRNA labeled with Cy3 using the Ambion MessageAmpII kit and 1.65µg labeled aRNA used to hybridize with each array
Hybridization protocol
Agilent one-color hybridization protocol. To minimize post-amplification biases, we randomized the position and order of hybridizations.
Scan protocol
Scanned at 10% laser power to avoid signal saturation
Agilent Feature Extraction software (GE1-v5_95_Feb07) used to calculate fluorescence values which were then log2-transformed and rank-normalized across 120 hybridizations
