The raw reads of mRNA-Seq following the embryonic development in medaka (Oryzias latipes)
Description
The samples of total RNA were extracted from medaka fertilized eggs at 0, 1, 3, 5, 7 dpf (days post fertilization), and paired-end reads mRNA sequence was performed. Medakas of these samples are Oryzias latipes and normal phenotype. day0_1.fastq.gz and day0_2.fastq.gz are obtained from the 0 dpf sample, day1_1.fastq.gz and day1_2.fastq.gz are from 1 dpf sample, day3_1.fastq.gz and day3_2.fastq.gz are from 3 dpf sample, day5_1.fastq.gz and day5_2.fastq.gz are from 5 dpf sample and day7_1.fastq.gz and day7_2.fastq.gz are from 7 dpf sample.
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Steps to reproduce
Medaka fish and embryo Embryos for this study were taken from 15 adult medaka fish (mating pair, 10 female and 5 male) of the strain bred in our laboratory. The mating pair adult fish were put together into a culture tank (60 cm × 30 cm × 30 cm) with artificial sea water (1‰ salinity, 45 l). The water temperature was 27 ± 1 ◦C, and the illumination cycle was light: dark = 14:10. The fish were fed with Artemia nauplii juveniles (24 h incubation) two times per day (10:00 and 17:00). Fertilized embryos were taken within 5 min of spawning from the medaka mating pairs, and fertilized healthy embryos were selected under an anatomical microscope (SZX12, Olympus, Tokyo). The selected embryos were sterilized in 0.9% H2O2 for 10 min to minimize the risk of fungal and bacterial infection (Marking et al., 1994; https://doi.org/10.1577/1548-8640(1994)056<0225:afseoa>2.3.co;2). After that, embryos were cleaned with and cultured in the embryo culture medium (ECM) solution (0.1% NaCl, 0.003% KCl, 0.004% CaCl2-H2O, 0.008% MgSO4, 0.0002% Methylene Blue, pH = 7.0). Embryos were cultured in a petri dish, and it was kept on a shaker (30 rpm) at 27 ± 1 °C. Each 10 embryos were randomly taken as a pooled sample at the 0, 1, 3, 5, 7 days post fertilization (dpf). Total-RNA extraction On the each dpf, 10 embryos were randomly selected and transferred into 1.5 ml tubes containing 500 µl trizol-1%SDS, and homogenized (vortex, 20 s). 0 dpf sample was treated within 30 min after fertilization. Samples were stored at −80 °C until RNA extraction. Total-RNA was extracted following the protocol developed by (Chomczynski and Sacchi, 1987; https://doi.org/10.1016/0003-2697(87)90021-2), and using RNeasy Mini Kit (Qiagen, USA). The mRNA sample quality analysis and mRNA sequencing The mRNA was purified from total RNA using the NEBNext Poly (A) mRNA Magnetic Isolation Module (New England Biolabs Inc., USA). The quality of mRNA samples was analyzed and a parameter called the RNA integrity number (RIN) was generated by a bioanalyzer (Agilent 2100 Bioanalyzer, Agilent Technologies, USA). Such mRNA samples can be trusted only if the RIN is higher than 8.0, the threshold for high quality of mRNA, and the mRNA RIN values of the samples were over 8.0. The mRNA sequencing was done using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs Inc.), the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1, New England Biolabs Inc.), and the Miseq Reagent Kit v3 (300 cycles) (Illumina, MS-102-3003, USA) following the manufacturers’ instructions.