SWB-S1-2 Oxford Nanopore WGS
Floral buds were collected for DNA extractions from plants that were grown in growth chambers in the following conditions: 16 hour day length, 22C day and 18C night temperatures, with 60% relative humidity, and a light intensity of 460mE. Nuclei were extracted from the floral bud tissue following Lu et al (88), with minor modifications. We ground 1-1.5 g of floral bud tissue into a coarse powder in liquid nitrogen. Ground tissue was resuspended in LB01 buffer (15 mM Tris pH 7.5, 2 mM EDTA (Ethylenediaminetetraacetic acid), 80 mM KCL, 20 mM NaCl, 15 mM 2-Mercaptoethanol, 0.15% Triton-X 100 and 0.5 mM Spermine) on ice with intermittent mixing. Tissue homogenate was then filtered through four-layers of miracloth (EMD Millipore Corp) followed by filtration through a 20 m cell strainer (pluriStrainer). Filtrate was carefully pipetted on top of equal volume Density Gradient Centrifugation Buffer (1.7 M Sucrose, 10 mM Tris-HCl pH 8.0. 2 mM MgCl2, 5 mM 2-Mercaptoethanol, 1 mM EDTA, 0.15% Triton-X 100) in a 50 ml falcon tube and centrifuged at 2500g for 30 mins at 4. After centrifugation, supernatant was decanted and the nuclei pellet was used to isolate high-molecular weight (HMW) DNA using the Nanobind Plant Nuclei Big DNA kit (Circulomics, Baltimore, MD, Cat # NB-900-801-01). Nanopore libraries were prepared using the Oxford Nanopore SQK-LSK110 Ligation Sequencing Kit following the manufacturers recommendation. Purified libraries were loaded onto a FLO-PRO002 (vR9.4.1) flow cell. We performed sequencing using an Oxford Nanopore Technologies PromethION sequencer (release 21.02.7) running for 96 hours. Resulting FAST5 files were basecalled using the Nanopore Guppy basecaller software (v4.3.4).