title: RADseq of Hyperolius molleri- Hyperolius molleri CAS251602
design: We used the double-digest RADseq laboratory protocol (ddRADseq; Peterson et al. 2012) to collect genome-wide SNP data. For each sample we digested 250-1000 ng of freshly extracted DNA with the restriction enzymes Sbf1 and MspI (New England Biolabs, Ipswich, Massachusetts), which have 8 bp (5'-CCTGCAGG-3') and 4 bp (5'-CCGG-3') recognition sites, respectively. DNA digests were purified with Agencourt AMPure beads prior to ligating barcoded Illumina adaptors to the fragments. We pooled equimolar quantities of each sample prior to size selection using a Blue Pippin Prep (fragment size range 430 – 530) and PCR-amplified the libraries with 12 cycles using proofreading Taq and Illumina’s indexed primers (all of which differed by at least two base pairs to reduce de-multiplexing errors). We sequenced two pooled libraries of 44 samples each on two lanes of Illumina HiSeq 2000 (100-bp, single end) at the Cornell University Genomics Facility.