normalization data transformation protocol
Raw data was extracted using the Agilent Feature Extraction Software (Version 10.7.3.1). Array quality was monitored using the Agilent QC tool with the metric set Ge2_107_Sep09. ID_REF = VALUE = Normalized log10 ratio Cy5/Cy3.
array scanning protocol
Scanned on an Agilent G2565AA microarray scanner at 5 µm resolution.
Hybridization was performed onto 4 x 44k microarray slides, containing oligonucleotide 60mers designed with the Agilent eArray online platform, using the Agilent Gene Expression Kit according to the manufacturer's protocol.
80 ng of total RNA were amplified, reverse transcribed and labelled using the Agilent Two-color Low Input Quick Amp Labeling Kit according to the manufacturer's protocol. As a minor modification, random hexamers with attached T7 promotor were added.
sample treatment protocol
F. vesiculosus pieces were or were not grazed by Idotea baltica for 15 and 18 days.
Fucus vesiculosus pieces were kept in 25L aquaria at a mean (± SD) seawater flow rate of 184 (± 17) ml min-1. Mean (± SD) water temperature was 19.7 (± 1.0) °C. Light was provided in a light/dark cycle of 12/12 h at a mean (± SD) photon flow rate of 121.1 (± 2.9) µmol m-2 s-1 (PAR) during light periods.
nucleic acid extraction protocol
Total RNA was isolated with using 1 ml of a CTAB extraction buffer (2% CTAB, 1 M NaCl, 100 mM Tris pH 8, 50 mM EDTA pH 8) and 25 µl DTT 2 M. After incubation at 45°C for 15 min, an equal volume of chloroform:isoamylalcohol (24:1) was added, vortexed vigorously for 5 min and afterwards centrifugated for 20 min at RT and 12,000 g. Subsequently, the aqueous phase was transferred and 0.3 volumes of EtOH 96% were added. After mixing gently, a second chloroform extraction was performed. Subsequently, total RNA was extracted using the Qiagen Plant Mini Kit accoding to the manufacturer's protocol.