title: ddRADSeq of Poecilimon orbelicus: muscle
design: We used Qiagen DNeasy kits (Qiagen, Hilden, Germany) to extract and purify genomic DNA from the hind femur of each individual. We processed genomic DNA into one genomic library (100 individuals/library) using the double-digestion restriction-site associated DNA sequencing procedure (ddRAD-seq) described in Peterson et al. (2012) (PLOS One, 7: e37135). In brief, we digested DNA with the restriction enzymes MseI and EcoRI (New England Biolabs, Ipswich, MA, USA) and ligated Illumina adaptors including unique 11-bp barcodes to the digested fragments of each individual. We pooled ligation products and size-selected them between 350-450 bp with a Pippin Prep machine (Sage Science, Beverly, MA, USA). We amplified the fragments by PCR with 12 cycles using the iProofTM High-Fidelity DNA Polymerase (BIO-RAD, Hercules, CA, USA) and sequenced the library in two single-read 150-bp lanes on an Illumina HiSeq2500 platform at The Centre for Applied Genomics (SickKids, Toronto, ON, Canada).