Illumina HiSeq 2500 paired end sequencing; Subtle effects of radiation on embryo development of the 3-spined stickleback
Subtle effects of radiation on embryo development of the 3-spined stickleback
Adult sticklebacks were kept in artificial reproduction conditions in order to generate the in vitro fertilized embryos. Four couples were used to generate the embryos. 104 embryos were used for next generation sequencing analyses. 3 replicates of pool of 4 to 10 embryos per condition were used for NGS analyses. Each embryo was placed in individual glass tube and waterborne exposed to 3 mL of a radioactive solution of 0.1, 1 and 10 mGy/day using 32P and artificial water. These doses encompass the chronic low (0.1 mGy/day) and medium (1 mGy/day) doses encountered in the environment at Chernobyl 30 years after the accident, and the initial high dose to fish after the accident (10 mGy/day). These doses span the protection threshold of ecosystems set at 10 uGy/h (0.24 mGy/day). Control embryos were kept in clean artificial water. The embryos were exposed for 10 days and euthanized according to schedule 1 of the Home Office Licence (ASPA, 1986) using tricaine methanesulfonate (Sigma).  Total RNAs were extracted using the High Pure RNA Tissue kit (Roche Diagnostics Ltd, West Sussex, U.K.) according to the supplier's instructions. RNA quality and integrity was evaluated using a bioanalyzer. RNA integrity numbers ranged from 7.4 to 9.5.  mRNA-seq libraries were prepared for each pool using the Illumina TruSeq mRNA library kit following standard protocols.