title: RAD-Seq of Rhegmatorhina: muscle tissue
design: To investigate nuclear genetic structure and allele frequencies for the largest possible sample of individuals across the hybrid zone, we collected RADseq (Baird et al. 2008) data from the 222 Rhegmatorhina individuals described in the Sampling section. Before preparing RADseq libraries, we evaluated the quality of DNA extracts by visualizing each on 1.5% agarose gels, and we considered samples having fragment lengths >1.5 kbp adequate for library preparation. We excluded extracts from 10 samples that fell below this size range (Table S1), and we constructed 3RAD (Bayona-Vsquez et al. 2019) libraries for the remaining 212 samples using XbaI and EcoRI-HF, with NheI to reduce formation of adapter-dimers. We cleaned ligation products using Sera-Mag Speed-Beads (Rohland and Reich 2012) at a ratio of 1.25:1 (v/v), and we created full-length libraries by amplifying the ligation products with 11 PCR cycles using iTru5 and iTru7 as described in Glenn et al. (2017). We quantified PCR-amplified libraries using a Qubit Fluorometer (Life Technologies, Inc.) and checked for valid constructs by visualizing each library on 1.5% agarose gels to ensure fragment distributions spanned 300-800 bp. After validation, we cleaned amplified libraries using Speed-Beads at a 1:1 ratio (v/v) and pooled all libraries. To reduce the total number of RADseq loci recovered, we had a commercial service (Georgia Genomics Facility, Athens, GA) divide the pool of libraries across four lanes of a Pippin Prep (Sage Science, Beverly, MA) and perform size selection using a 1.5% dye-free Marker K agarose gel cassette (CDF1510) set to capture fragments 550 bp +/-10%. After size selection, GGF staff combined all lanes of size-selected products and increased the concentration of size-selected libraries by performing six cycles of PCR recovery using standard Illumina P5 and P7 primers. GGF staff cleaned the resulting reaction product, quantified the size-selected pool using a commercial library quantification kit (F. Hoffmann-La Roche AG, Basel, Switzerland), and prepared a 10 M pool of libraries for paired-end (PE) 150 bp sequencing across two lanes of Illumina HiSeq 3000 sequencing at the Oklahoma Medical Research Foundation (Oklahoma City, OK).