RADseq of Fundulus-heteroclitus-UCD-BC-334-284
A custom DNA extraction method utilizing Agencourt Ampure XP beads (Beckman Coulter) was used to obtain genomic DNA for each individual. Briefly, fin clips were digested overnight at 56 C in 4.2 mg/ml Proteinase K followed by an Ampure XP bead cleanup and elution in low TE (10 mM TrisHCl, pH 7.5, 0.1 mM EDTA). Genotyping was performed using restriction site-associated DNA sequencing (RADseq) following the protocol developed by Ali et al. For each sample, 250 ng DNA was digested with the restriction enzyme SbfI. Individual barcodes were annealed to each sample by adding 2 ml indexed SbfI biotinylated RAD adapters (50 nM). These adapters feature 96 unique 8-bp barcodes that distinguish individuals within a single library. Libraries of 96 individuals were then pooled and fragmented to 200-500 bp using a Bioruptor NGS sonicator with 9 cycles 30 sec on/90 sec off. We assessed fragment size using a 2% sodium borate gel and further fragmentation was performed as needed. Dynabeads M-280 streptavidin magnetic beads (Life Technologies, 11205D) were used to physically isolate the RAD-tagged DNA fragments from off-target DNA and the final DNA was eluted in 55.5 ml low TE. These RAD libraries were then prepared for Illumina sequencing using a NEBNext Ultra DNA Library Prep Kit for Illumina. During this process, each RAD library of 96 individuals receives a unique barcode, which allows multiplexing of RAD libraries during sequencing.