Leaf samples from different positions on annually growing branches of Vitis vinifera L. plants were collected from field-grown plants, at the IMBB campus at 10 am. Some samples were store at -80oC, while others were used directly for DNA extraction.
DNA was extracted as described by Workman et al. (2018) with minor modifications using fresh leaves from the upper nodal positions of annually growing branches. One gram of fresh leaf-tissue from the uppermost nodal position was used for intact nuclei isolation. Leaf tissues were ground in liquid N2 with a mortar and pestle, for at least 30 mintransferred to a tube where 10 mL of fresh nuclear isolation buffer (NIB: prepared according to Workman et al. 2018) was added. Nuclei were isolated in 10 ml NIB. Tubes were agitated on a rotary platform at max speed for 15 min at 4 °C. Homogenates were filtrated through a strainer (40nm) and then through 5 layers of Miracloth into 50 mL tubes. Samples were centrifuged at 4 °C for 20 min at varying centrifugal forces (herein 2500g, 4000g, and 5000g). The supernatants were discarded and 1 mL cold NIB was added to each pellet, and then resuspended with a paintbrush presoaked in NIB; suspensions were transferred in a new tube. Volumes were adjusted to 15ml by ice-cold NIB and the samples were centrifuged at 5,000g, 2,500g or 4,000g for 15 min at 4 °C. This step was repeated 3-4 times until the supernatants were colourless. Next, pellets were resuspended in 1 mL 1X Homogenization Buffer (HB: prepared according to Workman et al. 2018) and transferred to 1.5ml microcentrifuge tubes. Samples were either snap frozen or used directly. For long term storage, samples were spun down at 2,500 g for 5 min, supernatants were discarded and pellets were snap-frozen in liquid N2 and stored at -80 °C. Nuclei were stained for 5 min with an aqueous solution of 1 μm DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride) and examined by Confocal Microscopy.
For confocal microscopy of nuclei, fluorescence images were captured on a Leica SP8 (Leica Camera AG, Wetzlar, Germany). The fluorescent images (micrographs) were acquired at RT with 40x objective (N.A.=1.2), pinhole adjusted to 1 airy unit, and mounting medium ddH2O. The excitation wavelength was 405nm (Emission 412nm-467nm) for DAPI.
Nanobind-assisted DNA purification
Pellets were resuspended in 60 μL of Proteinase Κ and vortexed, followed by addition of 20 μL RNase A and vortexed again until a homogenous mixture was obtained. Then, 140 μL Buffer PL1 was added and vortexed extensively. Thorough mixing at this step is necessary to ensure complete lysis of the nuclei. Samples appeared cloudy and very viscous but homogeneous and were incubated on an Eppendorf ThermoMixerTM C (Thermo Fisher Scientific Inc.), at 55 °C and 900 rpm for 30 min. Lysates were centrifuged for 5 min at 16,000g at RT. The supernatants were transferred to 1.5 mL Protein LoBind microcentrifuge tubes using a wide bore pipette. Nanobind disks were added to the supernatants followed by addition of a 1X volume of isopropanol. The samples were carefully mixed by manual inversion for 20 min at RT. The microtubes were placed in a magnetic tube rack as per instructions from the Nanobind Plant Nuclei Big DNA Kit. Supernatants were discarded, 500 μL of Buffer PW1 were added and samples were mixed 4X by inversion. This step was repeated twice then, any residual liquid from the tube caps was removed by spinning on a microcentrifuge for 2s. Finally 100-200 μL Buffer EB was added and the tubes were spun on a microcentrifuge for 2 s and incubated at RT for 10 min. The extracted DNA was collected by transferring the eluate to a 1.5 mL microcentrifuge tube using a wide bore pipette. Tubes were further spun on a microcentrifuge for 5 s and remaining eluted DNA was collected and transferred to the same 1.5 mL microcentrifuge tube. The quantity and the quality of the extracted DNA were assessed by NanoDrop (NanoDrop ND-1000 Spectrophotometer) and Qubit (Invitrogen Qubit 3’).
DNA Library construction For Flongle
According to the manufacturer, 3 to 20 fmoles of DNA are required for Flonge Cells. Libraries were prepared using the 1D ligation sequencing kit (SQK-LSK109). Briefly, the workflow consisted of first an end-repair/dA-tailing step to repair blunt ends and to add an “Adenine” to the 3’ end of the amplicon, followed by adapter and motor protein ligation onto the prepared ends using NEB ligase (New England Biolabs). Subsequently, a bead-based purification step was used to enrich the adapter-ligated fragments and remove excessive nucleotides and enzymes. Finally, the library mixture was loaded into the Flongle flow cell and the MinKNOW GUI (version 19.06.8) was used for data acquisition as per recommendation from ONT. Each sequencing experiment was run for 24 hours (>60 active pores) or after a minimum of 500 reads were acquired for downstream data analysis. Promega Biomath Calculator (https://worldwide.promega.com/resources/tools/biomath/) was used to determine the amount of DNA in fmoles, using the starting yield of nucleic acids and the estimation of N50 of the reads ca. at 30-40 kb. The final yield of the libraries varied between 3-5 fmoles, which is the minimum amount required. Therefore, for the construction of the second library (referred to as “Vitis 2”, 4,000g), the starting yield of DNA was increased 4x at 2.5μg. After the ligation with the reagents used in double quantities, the final yield was 0.63μg, with 25% recovery, and was loaded in the Flongle.
In Silico Data Analyses
Raw FAST5 files produced by MinION were base-called using the integrated live base-calling setting in the MinKNOW Core 3.6.5. and Guppy 3.2.10 (Wick, Judd and Holt, 2019) that converts Fast5 files to FastQ. To proceed with the analysis it is important to know that the quality baseline (Q) is 7 by default in MinION. The goal is to achieve Q ≥ 8.5 and increased mean read length and higher outcome. Control DNA was eliminated by NanoLyse [1.2.0] and served as quality control for each run using NanoPlot1.13.0, and in combination using NanoComp 0.16.4. These tools are integrated into the NanoPack suite (De Coster et al., 2018).