Cannabis sativa Purple Kush genomic DNA library CS-PK_SIL-C
Paired-end genomic DNA libraries were constructed using reagents from the NEBNext DNA Sample Prep Reagent Set 1 (New England Biolabs) or the Paired-End DNA Sample Prep Kit (Illumina). Genomic DNA (5-10 µg) was sheared using the Bioruptor Standard sonication device (Diagenode) for 20 min on low power using 30 s “ON” and “OFF” cycles. Fragmented DNA was purified using the Qiagen PCR purification kit, and was subjected to an end-repair reaction for 30 minutes at 20°C containing 1× end-repair buffer, 0.4 mM 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 reaction was then purified using the Qiagen PCR purification kit and the 3’-ends of the DNA were adenylated for 30 min at 37°C in a reaction containing 1× Klenow buffer, 0.2 mM dATP, and 15 U Klenow (Exo-) in a final volume of 50 ?l. Adenylated DNA was again purified using the Qiagen MinElute PCR purification kit. Adapters were ligated to the purified DNA for 30 min at 20°C in a reaction containing 1× T4 DNA ligase buffer with ATP, 0.3 µM Adapter Oligo Mix, and 2000 U T4 DNA Ligase in a final volume of 50 µl. Ligation reactions were immediately analyzed on a 1% agarose gel and bands of the desired size were excised and purified using the QIAQuick Gel Extraction Kit (Qiagen). 1-2 µl of the purified DNA was used in a PCR reaction containing 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 in a final reaction of 50 µl. Thermal cycler conditions were as follows: 98°C for 30 s, 10 cycles of 98°C for 15 s, 65°C for 30 s, and 72°C for 30 s, followed by 72°C for 5 min. PCR reactions were run on a 1% agarose gel and fragments of the desired size were excised and purified using the QIAQuick Gel Extraction Kit.