title: WGA of Solanum tuberosum: leaf
design: 3.5 l NEBNext FFPE DNA Repair buffer and 3.5 l Ultra II End-prep reaction buffer were added to the sample and mixed by slowly pipetting with a wide-orifice tip. 2 l NEBNext FFPE DNA Repair mix and 3 l Ultra II End-prep enzyme mix were added to the sample directly after buffer homogenization, mixed by slowly pipetting with a wide-orifice tip and incubated for 5 minutes at 20C followed by 5 minutes at 65C in a thermal cycler. The end-repaired sample was mixed with 60 l AmpureBeads and was placed for 15 minutes on a horizontal test tube rotator for DNA binding, subsequently placed on a magnetic stand for 2-3 minutes. After settling of the bead pellet, supernatant was discarded without disturbing the pellet and the samples washed twice with 200 l 70 % (v/v) high purity ethanol. Reelution was performed in 61 l nuclease-free ddH2O for 60 minutes at 37C in an overhead rotator. Adapter ligation was performed by addition of 25 l ONT library kit ligation buffer (LNB), 10 l NEBNext Quick T4 DNA ligase and 5 l ONT library kit adapter mix (AMX) to the remaining 60 l end-repaired hmDNA sample. The sample was mixed by slowly pipetting with a wide-orifice tip and was incubated for 10 minutes at 25C in a ThermoBlock. A second cleanup was performed by adding 40 l AmpureBeads to the sample followed by incubation for 15 minutes at RT on a horizontal test tube rotator. After discarding the supernatant, the bead pellet was washed twice with 250 l ONT library kit L Fragment buffer (LFB) by resuspending the beads through very careful flicking of the tube until beads were homogenously dissolved in the LFB. Re-elution of the sample was performed in 15 l ONT library kit elution buffer (EB) for 15-20 minutes at 37C on an overhead rotator. 12-14 l sample containing 150  500 ng hmDNA were mixed with 37.5 l ONT library kit sequencing buffer (SQB) and brought to a final volume of 75 l with nuclease-free ddH2O. The sample was mixed by slow pipetting using a wide-orifice tip. The total sample volume was added to the SpotON port of the FlowCell.
identifiers.namespace: SUB6512918
identifiers.id: MinIon_6