GSM7047212: Eggs, fertilized with UV irradiated sperm, rep6; Oncorhynchus mykiss; RNA-Seq
Eggs preserved in RNA-later solution were incubated overnight in a refrigerator (4 °C) and finally stored at −80 °C until RNA purification. Two to three eggs, depending on their size, were used for RNA isolation for the transcriptomic study. RNA was isolated as previously described (Ocalewicz et al. 2019) using modified TRIzol procedure established at the Igor Babiak laboratory, University of Nordland, Norway. The quality of the obtained RNA was established using TapeStation4150 (Agilent Technologies) and RNA ScreenTape Assay allowing RIN (RNA Integrity Number) evaluation. Good quality isolates (RIN>8) were quantified using Qubit™ RNA Assay Kits (Thermo Scientific) and 250 ng of a total RNA was used for library preparation with Illumina Stranded mRNA Prep kit (Illumina) and standard protocol. The final libraries were controlled for quality using D1000 Agilent ScreenTapes and TapeStation4150 system, and quantified using Qubit™ dsDNA BR Assay Kit (Thermo Scientyfic). Equimolar pool of libraries was finally commercially sequenced on NovaSeq6000 System (Illumina) in a paired-end read of 2x150 bp to obtain a high sequencing depth (i.e. minimum of 75M PE reads per sample).