Irys NLRS DNA labeling and Data collection
Equilibrate DNA ~30 min at room temp.
Pipet mix 3x with wide bore tip.
Prepare nicking master mix One 10μL rxn: 1μL buffer + DNA + enzyme in 200μL thin-wall PCR tube.
Blood BspQI: 5-7u (BNG3)Cell line BspQI: 3-5u (BNG3)Other BspQI: 5-10u (BNG3)BbvCI: 4-8u (BNG2)Q+C: 3/2, 6/4, 9/6u (BNG2). 
Mix 4x with Xplorer Plus Pipettor set to 8μL at lowest speed.
Pulse spin 2sec.
Incubate in a thermal cycler for 2 hrs at 37°C with heated-lid.
(For the rest of the protocol, protect from light)
Prepare the Labeling Master Mix.
reagentamount10x labeling buffer1.5μL10x labeling mix1.5μLTaq 5u/μL1μLH201μL.  
Add 5μL Labeling Master Mix.
Mix 4x with Xplorer Plus Pipettor set to 13μL at lowest speed.
Pulse spin 2sec.
Incubate in a thermal cycler for 60 min at 72°C with heated-lid.
Prepare the Repair Master Mix.
reagentamount10x Thermo Pol0.5μL50x Repair Mix0.4μL50mM NAD+0.4μLTaq DNA ligase1μLH202.7μL.  
Mix 4x with Xplorer Plus Pipettor set to 18μL at lowest speed.
Pulse spin 2sec.
Incubate in a thermal cycler for 30 min at 37°C with heated-lid.
Place NLR reaction on ice.
Add 1μL Stop Solution.
Mix by gently stirring 5 times with the pipet tip.
Pulse spin 2sec.
Add 5μl of the Repair Master Mix.
Equilibrate Staining Master Mix components to room temp.
Prepare the Staining Master Mix.
reagentamount4x flow buffer15μL1M DTT12μLDNA stain1.5μLH2O11.5μL.  
Aliquot 40μL of the Staining Master Mix into a 0.6mL amber tube.
Transfer NLR to Staining Mix with standard tip.
Pulse spin 2 sec.
Gently mix NLRS DNA 5x with wide bore tip set to 50μL.
Pulse spin 2 sec.
Place staining reactions at 4°C overnight to ensure uniform DNA staining.
Equilibrate NLRS to room temp for ~30 min.
Gently mix DNA 3x with wide bore tip set to 50μL.
Pulse spin 2 sec.
Remove 2μL aliquots from the Top, Middle and Bottom to separate Qubit Assay tubes.
Add 18uL of Qubit HS Buffer.
Sonicate 10 min in a bath sonicator.
Add 180μL Qubit reagent mix.
Vortex 5 seconds.
Pulse spin 2 sec.
Incubate at least 2 min at room temp.
Quantitate with Qubit 2.0 Fluorimeter.
NLRS DNA conc: 3-10ng/μL.
CV < 25%.
Load sample into Irys chip and start data collection with a 30-cycle run on irys system
