Bulk-RNA sequencing on Oryzias sakaizumii Kaga and Oryizas latipes Cab tails 13-14SS
Bulk-RNA sequencing on Oryzias sakaizumii Kaga and Oryizas latipes Cab tails 13-14SS
Standard conditions for fish breeding and egg collection. Medaka Oryzias latipes (Cab), Oryzias sakaizumii (Kaga) strains are maintained as closed stocks in a constant recirculating system at 27-28°C, with a 14hr light / 10hr dark cycle in the EMBL Laboratory Animal Resources (LAR) fish facility. Females and Males of each strain were put together and eggs were raised until the 13-14 SS. Tails at 13-14 SS were dissected and total RNA was extracted. Briefly, dechorionated Cab and Kaga embryos at the 12-13 somite stage were placed in Gibco CO2 independent medium (Thermo Fisher #18045054). Using a forceps and a scalpel, tails were cut directly at the unsegmented presomitic mesoderm (PSM) border. Dissected tails were then disrupted and homogenised for total RNA extraction using RNeasy Plus Micro Kit (Qiagen #74034) following the manufacturer's guidelines. The integrity and concentration of the extracted RNA was checked by using Agilent Bioanalyzer with the RNA 6000 Nano Assay kit. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) together with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) using the long inserts version of the manufacturer's protocol. Briefly, these modifications consisted of 7 minutes of fragmentation, 50 minutes extension for the first strand synthesis, 1:100 dilution of the adaptor and size selection for a 400 base pair long insert. The libraries were quantified using the Qubit HS DNA assay as per the manufacturer's protocol. For the measurement, 1ul of sample in 199ul of Qubit working solution was used. The quality and molarity of the libraries was assessed using Agilent Bioanalyzer with the DNA HS Assay kit as per the manufacturer's protocol. The assessed molarity was used to equimolar combine the individual libraries into one pool for sequencing.