title: ddRADseq of Eutagenia smyrnensis: adult whole organism
design: Individual was killed and preserved in 100% ethanol and stored at -20C. DNA extraction was performed following a commercial bead-based protocol (Biosprint 96 DNA Blood kit, Qiagen) as implemented in the automated KingFisher Flex system (Thermo Fisher scientific). Library preparation followed the double-digest Restriction site Associated DNA sequencing (ddRADseq) protocol of Peterson et al. (2012) (PLOS One, 7: e37135) with small modifications (Papadopoulou and Knowles, 2017; Evolution, 71: 2901-2917). Briefly, DNA was double-digested with restriction enzymes EcoRI and Msel, unique barcodes (10bp) and adaptors were ligated to the digested fragments and individually barcoded products were pooled into libraries of 72-80 samples each. Each library was size-selected between 350 to 450 bp using a Pippin Prep instrument (Sage Science Inc.). Following size-selection, the fragments were PCR-amplified (10 amplification cycles) using high-fidelity DNA polymerase (iProof , Bio-Rad) and they were sequenced on the Illumina HiSeq2500 platform (single-end, 150bp reads) at the Centre for Applied Genomics (SickKids, Toronto, Ontario, Canada).