title: RAD-Seq of Vespula pensylvanica: pupa gyne head
design: We extracted DNA from the head of each pupa using a modified version of the Qiagen DNEasy protocol for insect tissue (Qiagen, Inc). Deviations from the protocol include the substitution of 70% ethanol for AW2 buffer and the use of alternatively-sourced spin columns (bpi-tech.com). We constructed RADseq libraries using a customized protocol (Brelsford et al., 2016). In brief, we digested DNA with the enzymes SbfI and MseI, ligated adapters (including 96 unique in-line barcoded adapters to the SbfI end), removed small DNA fragments using Sera-Mag Speed Beads (Thermo Fisher Scientific), ran quadruplicate PCRs for each individual (adding a unique Illumina index sequence for each plate), pooled the four replicates and added additional primer mix and dNTP for one final PCR cycle to minimize single-stranded DNA fragments, ran each sample on a gel to ensure that the library was successful, and pooled individual libraries. We ran a final bead clean-up to remove small DNA fragments from the pooled library before sequencing.