title: ddRAD-seq of Formica cinerea: worker
design: We extracted DNA from the head and thorax of workers, and only the head of gynes and males. For the 2014 and 2018-2020 samples, we used the QIAGEN DNeasy Blood & Tissue Kit with modifications described in McGuire et al. 2021. Briefly, we manually ground the tissue with sterile pestles in a 1.7 ml tube while immersed in liquid nitrogen, and left the pulverized samples overnight in buffer ATL and proteinase K at 56C. We then transferred the supernatant to the QIAcube HT/QIAxtractor robot to complete the extraction. We eluted the DNA in 100 L of buffer EB. We sequenced all samples using a double-digest restriction site-associated DNA sequencing (RADseq) approach (protocol from Brelsford et al.2016). We digested the DNA using restriction enzymes MseI and PstI and ligated a universal MseI adapter and uniquely barcoded PstI adapter to each sample. We then removed small DNA fragments using Serapure magnetic beads or Omega magnetic beads (Omega Bio-tek, 2021) in a 0.8:1 ratio (beads: sample solution). We amplified each sample in four separate PCR reactions with indexed Illumina primers and then pooled the replicate PCR products for each sample for a final PCR cycle. Finally, we pooled all PCR products in a tube and did a final round of small fragment removal using the magnetic beads. Sample sizes in each batch are provided.