title: PacBio RS II Cabernet Sauvignon FPS 08
design: DNA Isolation    High quality genomic DNA was isolated from grape leaves (Cabernet Sauvignon clone 08). Plant tissue (1 g) was ground to a powder in a mortar containing liquid nitrogen. Ten mL of pre-warmed (65 oC) extraction buffer (300 mM Tris-HCl pH 8.0, 25 mM EDTA pH 8.0, 2 M NaCl, 2% (w/v) soluble PVP (MW 40000), 2% CTAB, 2% 2-mercaptoethanol) was added and the suspension was homogenized by inversion and incubated at 65 oC for 30 min in a water bath, mixing by inversion every 5 min during incubation. Sample was centrifuged at 5000 rpm for 5 min at room temperature and the supernatant was transferred into a new tube. Equal volume of chloroform:isoamyl alcohol (CIA, 24:1 v/v) was added and mixed by inversion for 5 min. Sample was centrifuged at 5000 rpm for 10 min at room temperature and the aqueous phase was transferred gently into a new tube. RNase A was added to the sample (2 µg) and was incubated at 37°C for 30 min. After RNAse treatment, equal volume of CIA was added and centrifuged as above. Aqueous phase was collected and 0.1 volume of 3 M NaOAc pH 5.2 and an equal volume of isopropanol were added to precipitate the DNA, sample was mixed by inversion and then incubated at – 80 oC for 30 min. Precipitated DNA was centrifuged at 5000 rpm for 30 min and supernatant was discarded. DNA pellet was washed twice with 3 mL of 70 % ethanol and centrifuged at 5000 rpm for 10 min. DNA pellet was air-dried at room temperature and resuspended in 500 µl of nuclease-free water. DNA quality was evaluated by pulse-gel electrophoresis, and quantity was determined using the Qubit fluorometer.    SMRTbell Libraries Preparation    Isolated genomic DNA was further cleaned with 0.45X AMPure PB beads (Pacific Biosciences) to ensure good purity ratios before library preparation. Three aliquots of gDNA (5 µg) were fragmented using the Megaruptor (UCDavis, Genome Center) for the 30 Kb shear. After shearing, sample was cleaned and concentrated using 0.45X AMPure PB beads. SMRTbell template was prepared with 6.1 µg of sheared DNA using SMRTbell Template Prep Kit (Pacific Biosciences) following the manufacturer’s instructions. 30 ul of SMRTbell template was loaded into the Sage Blue Pippin for size selection and a cutoff range of 12,000-50,000 bp was selected. Size selected library was cleaned with 1X AMPure PB beads and a repair DNA damage treatment was performed. After DNA repair, a new clean up step with 1X AMPure PB beads was done. Final amount of SMRTbell Library was 20.8 ng/µl in 10 µl (208 ng).