RAD-Seq of etheostoma caeruleum: adult
Total genomic DNA was extracted from fin clips using a DNeasy Blood and Tissue Kit, according to manufacturer protocols (QIAGEN, Hilden, Germany). After extraction, individual DNA concentration was quantified using either a Qubit 3.0 fluorimeter dsDNA HS Assay kit (Thermo Fisher Scientific, MA, USA) or by Quant-iT PicoGreen assay (Thermo Fisher Scientific, MA, USA). Library preparation followed a modified restriction site-associated DNA sequencing (RADseq) protocol described by Ali et al. (2016). Briefly, samples were divided into two libraries, with individuals concentrated to a target DNA of 120ng in 10l total volume. Samples were digested using the SbfI restriction enzyme and ligated with a unique, eight base pair length adapter sequence unique to each library. Samples were pooled by library and then sonicated to ~500 base pair fragments using a M220 Focused-ultrasonicator (Covaris, MA, USA). Sonicated DNA was ligated to Dynabead M-280 Streptavidin beads for a series of four washes, after which the DNA was liberated using the SbfI restriction enzyme. Preparation for sequencing followed the protocol outlined in the NEBNext Ultra DNA Library Prep Kit for Illumina with no modifications (New England Biolabs, MA, USA). Six base pair length adapter sequences were added to identify replicate individual adapter sequences between the two libraries. Agencourt AMPure XP (Beckman Coulter, IN, USA) bead size selection was performed based on the DNA concentration obtained from a Qubit 3.0 fluorimeter. The final libraries were submitted to the RTSF Genomics Core at Michigan State University and were pooled for sequencing in one lane with paired-end 150 base pair reads on an Illumina HiSeq 4000 (San Diego, CA, USA).