Cannabis sativa Purple Kush genomic DNA library CS-PK_2kb-2a
Mate pair libraries were prepared using the Mate Pair Library Preparation Kit v2 (Illumina Inc.). 10µg genomic DNA was fragmented using the Covaris S2 Adaptive Acoustic Device following the manufacturer’s recommendations. The fragmented DNA was subjected to an end-repair reaction for 30 min at 20°C containing 1× end-repair buffer, 0.15 mM dNTPs, 0.25 mM biotinylated dNTPs, 15 U T4 DNA Polymerase, 5 U Klenow Large Fragment, and 50 U T4 PNK in a final reaction volume of 100 µl. The DNA was then run on a 0.8% agarose gel and bands of the desired size were excised and purified using the QIAQuick Gel Extraction Kit. 600 ng of purified DNA was circularized overnight at 30°C in a reaction containing 1× Circularization Buffer and 13.4 µl Circularization Ligase in a total volume of 300 µl. The next day, 3 µl DNA exonuclease was added to the reaction and incubated for 20 min at 37°C. The circularized DNA was fragmented using the Bioruptor sonication device as described above. The fragmented DNA was then applied to DynaI magnetic M-280 beads (Invitrogen) and washed as recommended by the manufacturer to enrich for biotinylated DNA fragments. The fraction of DNA bound to the beads was subjected to end-repair, adenylation, and adapter ligation as described above, except that each step was followed by a bead wash instead of column purification. The beads were resuspended in 50 µl PCR mix (1× Phusion buffer, 0.2 mM dNTPs, 0.5 µM each of PCR primers PE 1.0 and 2.0 and 2 U Phusion DNA Polymerase). Thermal cycler conditions were as described above, except that 18 cycles were used. Size selection, gel purification, and quantification of libraries were as described above. Mated libraries were sequenced as 2× 42 nt reads on an Illumina HiSeq instrument.