title: RADseq for Rana boylii: HBS30449
design: DNA was extracted from tissues using a salt extraction protocol (Sambrook and Russell 2001). Dual-indexed sequencing libraries were prepared using the 3RAD protocol, a variant of the double-digest RADseq protocol (Peterson et al. 2012), with MspI as the common-cutting enzyme, SbfI as the rare-cutting enzyme, and ClaI as the dimer-cleaving enzyme (Hoffberg et al. 2016). Individual libraries were pooled at equimolar ratios and size selected in a single batch to between 500 and 700 bp using a Pippin Prep (Sage Science, Beverly, MA). Libraries were sequenced on two 100bp PE HiSeq 2000 lanes. However, the R2 failed on one of these lanes, so we retained only the R1s from both lanes to take advantage of the increased depth from two lanes of sequencing. For both lanes, custom sequencing primers were used that overlapped the restriction sites so that the first base of each read was the first base following the restriction site.