BioSample

Protocols: Gametes were obtained by dissection of four adults, and fertilization was performed in Petri glass dishes (diameter 4 cm). Embryos were reared in filtered seawater at 18°C. When samples reached the desired developmental stage (stages 27, 32 and 34 of Hotta), they were manually collected, pooled in groups of 200 individuals and immediately frozen in liquid nitrogen. The RNA samples were extracted from pool of 200 individuals by using the miRVana isolation kit according to the manufacturer’s protocol. Briefly, the mirVana™ miRNA Isolation Kit uses a rapid procedure to isolate small RNAs from tissue and cells using glass fiber filter (GFF)-based method. RNA purification procedures rely on organic extraction, followed by alcohol precipitation or adsorption of nucleic acid molecules on a GFF or silicate matrix. The quality of RNA was evaluated using the Tape Station 2200 instrument (Agilent). The RIN value ≥ 8 was taken threshold of good quality. The nucleic acid libraries were performed by using the SMARTer universal low RNA library (Ribo-Zero)plus rRNA depletion. To verify the size of PCR enriched fragments, the template size distribution was checked by by running on Agilent Technologies 2100 Bioanalyzer using a DNA 1000 chip. The Library QC analysis results in a concentration range from 30 t0 260 nM and an average size of 311 bp. To generate a standard curve of fluorescence readings and calculate the library sample concentration, Qubit standard Quantification solution and calculator was used.