Dneasy animal blood and tissue kit

Genomic DNA was extracted using the Qiagen DNeasy animal blood and tissue kit (Qiagen, Valencia, USA). The library was prepared using the double-digest RADseq protocol [35 (link)], with modifications (see electronic supplementary material, methods) and sequenced on a single Illumina HiSeq 2000 lane, at the UCLA Neuroscience Genomics Core facility. Raw data were de-multiplexed, quality filtered and trimmed to 95 bp, using the ‘process_rad_tags' script available in stacks v. 1.09 [36 (link)]. Loci were assembled using the stacks ‘de novo_map.pl' pipeline, while the ‘populations' script was used to filter loci and create output files (for raw data filtering and loci assembly see electronic supplementary material, methods). Loci were shared between the seven populations (p = 7), in at least 65% of individuals within a group (r = 0.65) and with a coverage of 8× (m = 8). We used only the first SNP of each sequence and removed loci with minor allele frequencies lower than 1.5% (i.e. at least two individuals must have the unique allele). Our quality control and filtering resulted in a total of 1174 loci and a data matrix that was 84% complete. We used pgdspider 2.0 [37 (link)] to convert the resulting structure files into other formats.

All DNA extractions were performed using the Qiagen DNeasy Animal Blood and Tissue Kit with slight modifications from the manufacturers’ suggested protocol. Whole blood or tail tissue sections in lysis buffer were thawed completely. One-half of the volume of blood (approximately 50 μl each whole blood and DNA lysis buffer) and approximately 100–200 mg of tail tissue in lysis buffer were combined with 40 μl Proteinase K and a variable volume of Buffer ATL for a total digestion volume of 400 μl. Following overnight digestion at 55°C with gentle shaking (~60 rpm), samples were combined with 400 μl Buffer AL and 400 μl 100% ethanol. Extractions then proceeded according to standard protocol, with elution in 200 μl AE repeated once for a total volume of 400 μl gDNA. Each batch of extractions included a single extraction blank (additional Buffer ATL instead of blood) that was carried through qPCR as well.