alias: ExSppsc104
title: UCE sequences of Exaerete smaragdina
design: DNA extractions were carried out using a standard phenol/chloroform protocol (http://www.danforthlab.entomology.cornell.edu/resources.html). Tissue samples were extracted from the specimens hind legs or thoracic muscles. We quantified DNA extracts using a Qubit 3.0 fluormeter (Thermo Fisher Scientific, Inc.) and assessed fragmentation by running 2 l on a 1.5% sodium borate gel. We sheared an average 215 ng DNA for each sample to a targeted fragment size of 500-600 bp using a Q800R sonicator (Qsonica, LLC.). First, we processed the samples that were generated with the enhanced HymV2-ant UCE bait set (Branstetter et al. 2017c; hereafter UCE v2). We followed the library preparation protocol of Faircloth et al. (2015), with modifications from Blaimer et al. (2016a; 2016b). We used a KAPA Hyper Prep Kit (Kapa Biosystems, Inc.), and prepared the libraries using Speedbeads (SPRI beads) for the cleanup steps (described in Fisher et al., 2011). We prepared dual-indexed libraries using TruSeq adapters following Faircloth and Glenn (2012). The PCR reagents, cycling parameters, and subsequent purifications correspond to Blaimer et al. (2016a; 2016b). Target enrichment was carried out for 21 samples with the UCE v2 bait set (Supplementary Table 1). The custom-made biotinylated RNA probes were purchased from MYcroarray, Inc. Hybridization reactions were run on a thermal cycler at 65C for 24h. Products were washed following Blumenstiel et al. (2010), and the post-enrichment PCR was conducted following Blaimer et al. (2016a; 2016b). Enrichment success was quantified using a ViiA7 (Thermo Fisher Scientific, Inc.) qPCR system and a KAPA Sybr Fast qPCR kit (Kapa Biosystems, Inc.). We set up a series of four dilutions according to Blaimer et al. (2016a), and inferred the input concentration by considering an average fragment length of 600 bp. Based on the inferred concentration for each library, we pooled the samples at equimolar concentrations. Lastly, we used a BluePippin device (Sage Science, Inc.) to select for fragment sizes between 250 and 800 bp length. Sequencing was carried out at the Cornell Biotechnology Resource Center (http://www.biotech.cornell.edu/biotechnology-resource-center-brc), using an Illumina HiSeq 2500 platform with 150 bp paired end reads. The newly generated UCE sequences that were captured using the initial UCE bait set (hereafter UCE v1) were processed by Rapid Genomics LLC., following the complete workflow outlined in Faircloth et al. (2015). The sequencing for this data (UCE v1) was carried out on an Illumina HiSeq 3000 platform with 150 bp paired end reads.This specific sample was generated using the UCE v2 bait set.
identifiers.namespace: SUB4560723
identifiers.id: ExSppsc104