2b-rad tags of ussuri catfish: adult muscle
Briefly, the restriction enzyme BcgI was used to digest genomic DNA of each individual. The adapter 1 and adapter 2 were ligated. Then a set of four primers that introduce individual specific barcodes and sequencing primers were applied to amplify ligation products using Phusion high-fidelity DNA polymerase. The amplification system had a volume of 25 L in total, containing 5 L of ligated DNA, 0.5 mM of P5 and P7 primers, 0.5 mM of P4 and P6-barcode primers, 0.75 mM of dNTPs (Sangon, China), 5 L of 5Phusion HF Buffer and 0.5 L of Phusion DNA polymerase. PCR reactions were performed on a 96-well thermal cycler. The products were separated in 8% polyacrylamide gels by electrophoresis, after purifications, equal amount of each product was mixed to make a final library.