High-Quality Nuclear DNA Extraction and Sequencing from N. benthamiana

The N. benthamiana used throughout this study is from an accession called ‘LabBenth’ that is routinely used at CSIRO, University of Sydney and New Zealand Institute of Plant and Food Research and available upon request. The nuclei were isolated as previously described [29] , with the following modifications: 20 g of fully developed leaves were homogenized in a kitchen blender at maximum speed for 30 s in 300 mL ice cold nuclei isolation buffer (0.5 M mannitol, 10 mM PIPES, 10 mM MgCl₂, 5 mM β-mercaptoethanol 2%, 10 mM sodium metabisulfite, polyvinylpyrrolidone (MW 40,000), 200 mM Lysine, 6 mM EGTA, pH 6). The homogenate was filtered through 4 layers of cheesecloth and 4 layers of miracloth. The filtrate was lysed by the gradual addition of Triton X-100 to a final concentration of 0.5% and the nuclei-enriched fraction collected by centrifugation (4°C; 15 min at 4967 g) using disposable 50 mL conical tubes. The pellets containing the nuclei were gently resuspended in the same volume of ice cold nuclei isolation buffer without β-mercaptoethanol, and centrifuged again. All pellets were resuspended and combined into 50 mL of the wash buffer and centrifuged again. The pellet was stored at −80°C prior to DNA extraction. The nuclear genomic DNA was extracted using a method originally optimised for RNA extraction from mango mesocarp [30] with some modifications as described in Hilario et al. [31] (link). The RNaseA treatment was performed during the lysis step and the phenol:chloroform extraction was omitted. The nuclear genomic DNA pellet was resuspended in 500 µL 10 mM Tris, 1 mM EDTA, pH 8 and centrifuged at 10000 g for 30 min to remove remnant starch granules. The starch-free supernatant containing nuclear genomic DNA was quantified by spectrophotometry at 260, 280 and 230 nm and stored at 4°C. A sample of DNA was analysed by pulse field gel electrophoresis to estimate the fragment size of the extracted sample at greater than ≥40 kbp.
Nuclear genomic DNA was sheared to an insert size of either ∼180 bp or ∼500 bp (Table 1) and prepared for paired-end sequencing on the Illumina HiSeq2000™ platform according to the manufacturer’s instructions. A LIMP (long insert matepaired end library) was also prepared according to manufacturer’s instructions and subsequently sequenced on one lane of the HiSeq2000™ platform. This library was subsequently analysed to contain ∼2000 bp inserts.