DNA-seq of adult Dinothrombium tinctorium: Insert size 3000bp
The mate pair library was constructed using Nextera Mate Pair kit from Illumina. 3 ŚĚg DNA was tagmented as described in the protocol and cleaned using Zymo Genomic DNA Clean & Concentratorţ column. The sample was then subjected to strand displacement and cleaned with AMPure XP beads. A 0.6% megabase agarose gel (BioRad) was used to separate out the fragments. The 2-5kb range of sized fragments were extracted and the fragments recovered using Zymocleanţ Large Fragment DNA Recovery Kit. The quantity of DNA recovered was measured and used in a circularisation reaction which required incubation at 16 degrees overnight. After purification with Ampure, DNA was sonicated to approx. 500bp with the Covaris and recovered with AMPure beads as before. Samples were bound to Dynabeads M280 and all subsequent reactions (end repair, A-tailing, adapter ligation) were bead-based. Samples were amplified with 10 cycles PCR, recovered by Ampure XP 1:1 and quantified using Qubit High sensitivity DS DNA kit.