Primescript 1st strand cdna synthesis

Total RNA was extracted from the brain and pituitary gland of each fish by using RiboEx LS^TM reagent (GeneAll Biotechnology Co., Ltd., Seoul, Republic of Korea) and quantified with a NanoVue spectrophotometer (GE Healthcare Life Sciences, Buckinghamshire, UK). For cDNA synthesis, the total RNA from the tissue was treated with DNase by using an RQ1 RNase-Free DNase kit (Promega Corporation, Madison, WI, USA) and reverse-transcribed by using PrimeScript^TM 1st-strand cDNA synthesis (Takara Bio., Inc., Otsu, Shiga, Japan). In detail, 1 μg of RNA from the brain or 200 ng from the pituitary gland was diluted with RNase-free water to a total of 8 µL in the PCR tube. This mixture was reacted with 1 µL of random hexamer primer (50 µM) and 1 µL of dNTP mixture (10 mM each), followed by incubation at 65 °C for 5 min. Subsequently, 10 µL of this mixture was added with 4 µL of 5X PrimeScript Buffer, 0.5 µL of RNase Inhibitor (40 U/µL), 1 µL of PrimeScript RT (200 U/µL), and 4.5 µL of RNase-free water. A total of 20 µL of mixture was incubated at 30 °C for 10 min, 42 °C for 60 min, and 95 °C for 5 min.

A batch of aE11 expressing hybridoma cells were sent to Genscript (GenScript Biotech [Netherlands] B.V.) for commercial Antibody Variable Domain Sequencing following their standard operating procedures, including lysis by TRIzol® Reagent (Ambion), PrimeScriptTM 1st Strand cDNA Synthesis (Takara), sequence retrieval by RACE, cloning and sequencing. A final report was provided containing the consensus variable gene sequences with corresponding V-gene and allele retrieved by IMGT-assisted analysis of V(D)J junctions.