Lucanus ML ddRAD
We used two restriction enzymes, EcoRI and MseI. The total amount of genomic DNA used for each individual was around 200 ng. T4 ligase was used for ligating barcodes. The barcoded DNA samples were individually purified using magnetic beads (Beckman Coulter). Polymerase chain reactions (PCR) with 8 cycles of reactions were applied to the cleaned-up fragments to attach the Illumina sequencing adaptors. The PCR products were individually cleaned-up using magnetic beads again and then pooled according to their DNA concentrations. We selected DNA fragments that were between 300 and 400 base pairs (bp) in size using Pipin Prep (Sage Science) before the Illumina sequencing. Single-end sequencing of 150-bp lengths was performed.