title: UCE target enrichment of Cethegus-sp-SDSU_MY0845: leg tissue
design: Library preparation followed Starrett et al. (2017), with some modifications. Briefly, libraries were prepared using the KAPA Hyper Prep Kit (Kapa Biosystems), using up to 250 ng DNA as starting material. Ampure XP beads (Beckman Coulter) were used for all cleanup steps. For samples containing less than 250 ng total, all DNA was used in library preparation. After end-repair and A-tailing, universal adapters were ligated onto libraries. Libraries were then amplified in a 25  l reaction, with 15  l adapter-ligated DNA, 25  l 1X HiFi HotStart ReadyMix, and 2.5  l of each Illumina TruSeq dual-indexed primer (i5 and i7) with modi ed 8-bp indexes. Amplification conditions were 98  C for 45 s, then 16 or 18 cycles of 98  C for 15 s, 60  C for 30 s, and 72  C for 60 s, followed by a final extension of 72  C for 60 s. Samples were quantified again to ensure amplification success. Equimolar amounts of libraries were combined into 1000 ng total pools consisting of eight samples each (125 ng per sample). Target enrichment was performed on pooled libraries using the MYbaits Arachnida 1.1K version 1 kit (MYcroarray, Inc.) following the Target Enrichment of Illumina Libraries v. 1.5 protocol (http://ultraconserved.org/#protocols). Hybridization was conducted at 60 or 65 C for 24 hours. Following hybridization, pools were amplified in a 50  l reaction consisting of 15  l of hybridized pools, 25  l Kapa HiFi HotStart ReadyMix, 5  l dH20, and 5  M of each of TruSeq forward and reverse primers. Amplification conditions consisted of 98  C for 45 s, then 16 or 18 cycles of 98  C for 15 s, 60 C for 30 s, and 72  C for 60 s, followed by a final extension of 72  C for 5 minutes.