DNA-Seq of Nephila clavipes: long insert (9 kb) mate-pair library of genomic DNA
Long insert, paired-end DNA "MPv2" mate pair libraries were constructed using Mate Pair v2 kits following the Ňgel-plusÓ protocol (Illumina) modified to include TruSeq DNA barcoded adapters (for multiplexing). Genomic DNA was sheared to fragments ranging from 1 kb to 25 kb using a Covaris S2 ultra-sonicator with red miniTUBEs, and subsequently end-repaired, biotinylated, and purified using AMPure XP beads at a 0.5:1 ratio. Fragments of sheared gDNA were then separated via gel electrophoresis (BioRad Certified Megabase agarose), excised in slices ranging from 8-10 kb (Ň9 kbÓ), and extracted with Zymoclean Large Fragment DNA Recovery Kits (Zymo Research). Extracts were circularized in 600 ng aliquots by incubating at 28ĄC for 16 hours, and subjected to exonuclease-treatments to remove linear gDNA. Circularized long-insert gDNA was then sheared to ~300 bp with a Covaris S2 focused ultra-sonicator with clear microTUBEs, then bound to M-280 streptavidin Dynabeads (Life Technologies) and repeatedly washed to remove non-biotinylated fragments. The remaining captured fragments were then end-repaired, adenylated, and ligated to TruSeq barcoded adapters using reagents from TruSeq LT kits (Illumina), then PCR amplified for 15 cycles using TruSeq PCR reagents, recipes, and cycling parameters. After PCR enrichment, final MPv2 jumping libraries were cleaned using AMPure XP beads at a 0.8:1 ratio.