Spawning of females was induced by thermal stimulation, consisting of exposure to alternate cycles of 29°C (1 hour) and 5°C (30 minutes). As each female began to spawn, it was removed from the spawning tank and transferred to an individual spawning beaker with filtered seawater at the same temperature. Once spawning was completed, the obtained oocytes were gently washed into a clean glass. The remaining oocytes of each female spawning were mixed with a sperm suspension (from 7 males) by gentle agitation, aiming to obtain around 10 spermatozoids by oocyte in a microscopic view. Moreover, to evaluate the quality of the collected oocytes, the D-larval rate (ratio between the number of free swimming larvae at 48h post fertilization and the number of starting eggs) of each eggs batch was registered. Based on that observation, oocytes were divided into two experimental groups: spawned oocytes with low hatching rate (LHR, 5%-21% of D-larval rate) and spawned oocytes with medium hatching rate (MHR, 40%-47% of D-larval rate). In addition, gonads from five females were dissected and oocytes were collected through a practice known as gamete stripping. As the name indicates, this procedure involves removal of gametes from gonad tissue. Briefly, fully ripe gonads were slashed repeatedly with a scalpel and washed with filtered seawater to harvest the gametes. Finally, sex determination and oocytes appearance were achieved through microscopy examination. About 20,000 oocytes for each spawning/stripping were collected and filtered in a 40 µm sieve. The oocytes were transferred into an Eppendorf tube and, after a short spin, the seawater was removed. To remove salts, the pellet of oocytes was re-suspended with a solution of ammonium formate 3%, which was immediately removed after a short spin. Then the oocytes were included in 1.5 ml of Extract-all solution (Eurobio) and preserved in liquid nitrogen until RNA isolation.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the Extract-all (Eurobio) procedure. RNA quality and integrity were controlled on the Agilent Bioanalyzer using RNA nanochips and Agilent RNA 6000 nanoreagents (Agilent Technologies, Waldbronn, Germany). RNA concentrations were measured at 260 nm using a ND-1000 spectrophotometer (Nanodrop Technologies) using the conversion factor 1 OD = 40 mg/mL total RNA. Samples were stored at -80°C until further use.
Label
Cy3
Label protocol
Amplified and labeled according to the Agilent Low Input One-Color Microarray-Based Gene Expression Analysis protocol.
Hybridization protocol
Hybridized to the Agilent-043932 array according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol.
Scan protocol
Array was scanned with an Agilent scanner G2565BA according to the standard Agilent protocol at a resolution of 2 microns modifying default setting to scan the slide twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%). The scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction v10.7.3.1 used XDR ID to link the pairs of scans together automatically when extracting data.
Description
3 Clam (Ruditapes decussatus) spawned between April 2013 and June 2013 in the experimental bivalve hatchery in Portugal. Oocytes RNA of Ruditapes decussatus.
Data processing
The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal. The fluorescence values were normalized by performing a quantile normalization in R statistical software. Statistical analyses were performed on 31,862 out of 59,951 probes with signal higher than background in at least 5 out of 15 target samples. A log base 2 transformation was applied to all expression values and finally, the parametric Combat algorithm was implemented in R in order to adjust for the known between-experiments batch effect.
Log2 normalized signal intensity ('null' corresponds to control probes; blank corresponds to probes with fluorescence values lower than the background in at least 5 out of 15 target samples)
