PacBio SMRT-Seq of Nephila clavipes: pooled LR-PCR amplicons of putative spidroin-coding genes
Long Range PCR (LR-PCR) primers were designed to amplify DNA encompassing each putative spidroin locus (from 2 kb to >25 kb in length) as identified from the Nephila clavipes draft genome. LR-PCR reactions employed high efficiency PrimeStar GXL polymerase (Clontech/TaKaRa) using manufacturerŐs recommendations and visualized on 0.5% Bio-Rad Certified Megabase agarose gels. Following LR-PCR, amplicons were cleaned using AMPure XP beads at a 1:1 ratio. Amplicons were pooled at equimolar ratios, with slightly higher volumes for the longest fragments (>20 kb). SMRT DNA library preparation and sequencing was performed using the P6-C4 sequencing enzyme, chemistry, and 4-hour movie collection parameters (Pacific Biosciences). Each spidroin amplicon pool was re-purified using AMPure XP beads at a 1.8:1 ratio. Each unsheared amplicon pool was end-repaired, incubated, and re-purified using AMPure XP beads at a 1.8:1 ratio. Blunt and SMRTbell adapters (Pacific Biosciences) were ligated to the DNA amplicons, which were then treated with an exonuclease cocktail to remove un-ligated DNA fragments, and final libraries were purified twice more using AMPure XP beads at a 1.8:1 ratio.