RADseq of Atelopus
We prepared libraries using a modified BestRad protocol (Ali et al., 2016). We started libraries with 200ng total DNA in 10ul sample volume for each sample. We digested sample inputs using the SbfI restriction enzyme and ligated samples with a unique, eight base pair length adapter sequence. We pooled samples and sonicated to 500 base pair fragments using a M220 Focused-ultrasonicatorTm (Covaris). We ligated DNA products to Dynabead M-280 Streptavidin beads for a series of four washes, after which we liberated the DNA using the SbfI restriction enzyme. We prepared libraries for sequencing using the NEBNext Ultra DNA Library Prep Kit for Illumina to manufacturers protocols (New England Biolabs). We purified product with Agencourt AMPure XP beads (Beckman Coulter) without size selection based on DNA concentrations. We sequenced our library in one lane with paired-end 150 base pair reads on an Illumina HiSeq 4000 at the Genomics Core at Michigan State University.