title: F0_Ovary.N2
design: Tissues were dissected from randomly selected female fish. Three ovaries per treatment group were used to prepare one pooled RNA sample. Two experimental replicates per normoxia and hypoxia treatment were prepared. Total RNA from pooled tissue samples was extracted using the mirVanaTM miRNA isolation kit (Applied Biosystems)  Small RNA (cDNA) library were constructed using TruSeq Small RNA Sample Prep Kit (Illumina), each prepared from 1 µg of total RNA. The cDNA libraries were prepared according to manufacturer’s instruction. Briefly, RNA 3' adapter and RNA 5' RNA adapters were ligated to each end of the small RNA molecule. The ligation products were used as a template for cDNA synthesis using SuperScript II Reverse Transcriptase (Invitrogen) to create single stranded cDNA. The cDNA was then PCR amplified using a common primer and a primer containing index sequences. After RT-PCR amplification, the cDNA libraries were purified on Novex 8% TBE polyacrylamide gels (Invitrogen) to select the libraries containing mature miRNA and other regulatory small RNA. The band containing the approximately 22 nt RNA fragment with both adapters were a total of 147 nt in length. The band containing the 30 nt RNA fragment with both adapters were 157 nt in length. The bands were extracted from the gel and purified using the MinElute Gel Extraction Kit (Qiagen). The sizes of selected library were validated by Agilent Technologies 2100 Bioanalyzer using High Sensitivity DNA chip.