ddRADseq of anolis sagrei: adult liver tissue
Whole specimens were formalin-fixed and livers dissected and preserved in 95% ethanol for DNA extractions. DNA was extracted from all tissues using a Qiagen DNeasy Blood and Tissue Kit (Qiagen, Inc.) and quantified using a Qubit dsDNA BR assay kit (Thermo Fisher, Waltham, MA). We prepared two genomic libraries following a modified double-digest RADseq (ddRAD) protocol of Peterson et al. (2012) (protocol in Barley et al. 2019): we digested 500 ng of each sample for 4 hours using the enzymes Sbfl and NlaIII. After ligation of the illumina adaptors, we pooled equimolar amounts of each samples that each had different adapter sequences and used AmpureX beads to purify the samples. We then quantified these using the Qubit dsDNA BR assay kit and and size selected for fragments between 375 and 475 bp using a Pippin Prep. We then ran PCR for 8-10 cycles using taq and the Illumina index primers for final library amplification. We quantified the final library concentration using the Qubit dsDNA HS assay kit. Prepared libraries were sent to the Oklahoma Medical Research Foundation at for sequencing on the NovaSeq platform, with one library per lane.