title: miRNA-Seq of Gobiocypris rarus: male rare minnows after 14days TPHP exposure
design: After 14 days exposure, triplicate samples (each sample 4 fish) in each treatment group were randomly removed and anesthetized with Tricaine (MS-222) before sectioning. Fish was dissected and liver tissues (~30 mg) were immersed in liquid nitrogen immediately after collection and stored at -80 oC. Then miRNA library was constructed, and an equal quantity (10  g) of total RNA was extracted from each of the various tissue samples. Briefly, 15  35 nt molecules were purified from total RNA (mir- Vana  microRNA Isolation Kit, Ambion, USA) using polyacrylamide gel electrophoresis (PAGE); then, 5  and 3  adaptors (TruSeq  Small RNA Sample Preparation Kit, Illumina, USA) were ligated to the 5  and 3  termini of the RNAs, and the samples were used as templates for cDNA synthesis. Subsequently, PCR amplification was performed using primers that were complementary to both adaptors. After purification of the amplified cDNA constructs, the products were sequenced using HiSeq2500 technology.