Species: Atlantic silverside (Menidia menidia)
Source: From laboratory breeding experiment or wild caught at one of four locations along the east coast of North America.
Library preparation method: Modified version of Illumina Nextera sample preparation kit.
Sequencing instrument: Illumina HiSeq 2000
Read type: Paired-end 125 bp
Sequencing strategy: Whole genome sequencing

The sequence data were prepared as described in Therkildsen & Palumbi (2017).

The tissue samples used for sequencing had been stored in minus 20 degrees C freezer for 8-17 years prior to DNA extraction. We used the Qiagen DNeasy Blood and Tissue kit to extract DNA from muscle tissue and evaluated the degradation level of each extract through 1.5% agarose gel electrophoresis. Only samples that showed clear high molecular weight bands and limited smearing were retained for library preparation. To ensure DNA integrity in the retained samples, we removed fragments shorter than ~1000 bp from each extract using Agencourt AMPure XP beads in a 0.4:1 AMPure to sample ratio and eluted the DNA in 10 mM Tris-Cl, pH 8.5.

We measured DNA concentrations with a Quant-iT high-sensitivity assay (Invitrogen) and prepared a separate barcoded library for each individual with Illumina’s Nextera kit according to the protocol described in Therkildsen and Palumbi (2017). Briefly, the tagmentation reaction, which simultaneously fragments the DNA and incorporates partial adapters, was carried out in a 2.5 ul volume with 1.6–7.9 ng of input DNA for each library. We then used a two-step PCR procedure with a total of 12 cycles (8 + 4) to add the remaining Illumina adapter sequence with dual index barcodes and amplify the libraries. The PCR was conducted with the KAPA Library Amplification Kit and the Illumina Nextera index kit with primers N501– N508 + S511 and N701–N712 + N714. As a final step, we purified and size-selected the amplification products with Agencourt AMPure XP beads and quantified the concentration of the final libraries with the Invitrogen Quant-iT high-sensitivity assay. We also examined the fragment size distribution of multiple libraries from each plate on an Agilent Bioanalyzer instrument.

We combined equimolar amounts of 56–76 libraries into separate pools for sequencing across 13.5 lanes of paired-end 125-bp reads on an Illumina HiSeq 2000 (v4 chemistry) at the University of Utah’s Bioinformatics Core Facility. To even out the data yield among samples, we repooled libraries that initially had obtained the lowest read output for supplementary sequencing in 4.5 additional HiSeq lanes.