DNA-Seq of Nephila clavipes: long insert (9 kb) mate-pair library of genomic DNA
Long insert, paired-end DNA "Nextera" mate pair libraries were constructed using Nextera Mate Pair library kits following the Revision D Ňgel-plusÓ protocol (Illumina) and feature transposome-mediated fragmentation and adapter tagging of circularized gDNA to facilitate identification of mate pair junction sequences, and include barcoded TruSeq adapters and TruSeq PCR reagents to enable multiplex sequencing. Genomic DNA was used as input for the tagmentation reaction,which replaces the initial sonication steps in the MPv2 protocol with transposome mediated gDNA fragmentation and the attachment of junction adapters. Tagmentation reactions were incubated then purified using a Genomic DNA Clean & Concentrator-10 Kit (Zymo Research) before undergoing strand displacement reaction. Fragments of gDNA were then separated via gel electrophoresis (BioRad Certified Megabase agarose and excised in slices ranging from 8-10 kb (Ň9 kbÓ), and extracted with Zymoclean Large Fragment DNA Recovery Kit (Zymo Research). Extracts were circularized by incubating at 30ĄC for 15 hours, then subjected to exonuclease-treatments to remove linear gDNA. Circularized long-insert gDNA was then sheared to ~300 bp with a Covaris S220 focused ultra-sonicator inclear AFA fiber tubes, 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 Nextera Mate Pair reagents and recipes (Illumina), then PCR amplified for 15 cycles using TruSeq PCR reagents, recipes, and cycling parameters (Illumina). After PCR enrichment, final Nextera jumping libraries were cleaned using AMPure XP beads at a 0.67:1 ratio.