Six hundred two pink-eye stage embryos, i.e., the late stage of the kairomone-sensitive period, were removed from maternal brood chambers and treated with either fed or starved Chaoborus-conditioned media. After incubation at 20°C for 1 hr, about half the animals in each treatment culture (150 embryos each) were collected and total RNA was extracted. After 4 hr additional incubation, the remaining animals were collected and total RNA was extracted. This procedure was repeated three times independently, resulting in 12 samples (4 conditions x 3 replicates)
Growth protocol
The clone of D. pulex used in the experiments was collected from a pool at Maeda Forest Park in Sapporo in 2009. The clone was reared in the laboratory at 20°C in aged tap water and fed unicellular green algae (Chlorella Industry Co. Ltd, Fukuoka, Japan) over generations in a temperature- and photocycle-controlled incubator (20°C, 16-h light/8-h dark)
Extracted molecule
total RNA
Extraction protocol
RNAqueous®-Micro Kit (Life Technologies, Gaithersburg, MD, USA)
Label
Cy3
Label protocol
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scan protocol
The slide was scanned by a microarray scanner G2565CA (Agilent Technologies, Santa Clara, CA, USA). To increase the accuracy of signal measurements by the scanner, we used two different photo-multiplier tube laser power settings (10% and 100%) for the target signal.
Data processing
Raw data were extracted as pair files using NimbleScan software version 2.6 (Roche NimbleGen) and combined to obtain a raw expression value for each probe (Dudley et al., 2002, Proc Natl Acad Sci U S A; Sato et al., 2007, Plant J).
