TITLE: Modified 1D Native Barcoding genomic DNA protocol  from the Temperton Lab (University of Exeter)
AUTHORS: Michael Henson, Ben Temperton, Cameron Thrash

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Modified 1D Native Barcoding genomic DNA protocol (v. NBE_9006_v103_revO_21Dec2016 for SQK-LSK108) from the Temperton Lab (University of Exeter)</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [DNA Fragmentation]
Load 46 μL of genomic DNA into a Covaris g-TUBE.
?. [DNA Fragmentation]
Spin the g-TUBE for 1 minute in an Eppendorf 5424 centrifuge.
?. [DNA Fragmentation]
Invert the tube and spin again for 1 minute.
?. [DNA Fragmentation]
Transfer volume to sterile 1.5 mL Eppendorf DNA LoBind tubes.     *Prior to the next step be sure to analyze your DNA for size, quantity, and quality.
?. [Library Preparation]
Combine: 45 μL genomic DNA, 7 μL Ultra II End-prep reaction buffer, 3 μL Ultra II End-prep enzyme mix, 5 μL of Nuclease-free water.
?. [Library Preparation]
Mix tube gently by flicking, then spin down.
?. [Library Preparation]
Transfer volume to a new 0.2 mL PCR tube.
?. [Library Preparation]
Incubate for 30 minutes at 20°C and 30 minutes at 65°C.
?. [Library Preparation]
Add 60 μL of AmPure XP beads and mix by pipetting.
?. [Library Preparation]
Incubate for 5 minutes at room temperature (RT), while flicking occasionally.
?. [Library Preparation]
Spin down the sample and pellet on the magnet.
?. [Library Preparation]
Pipette off supernatant while on the magnet, avoiding pellet.
?. [Library Preparation]
Wash beads with 200 μL of 80% ethanol (EtOH). Do not disturb the pellet. Remove EtOH.
?. [Library Preparation]
Repeat step 13
?. [Library Preparation]
Spin down the tube and place the tube back in the magnetic rack.
?. [Library Preparation]
Remove any residual ethanol.
?. [Library Preparation]
Allow beads to briefly dry.
?. [Library Preparation]
Remove the tube from the magnetic rack, and resuspend in 25 μL of prewarmed 55°C nuclease-free water.
?. [Library Preparation]
Incubate for 2 minutes at 55°C.
?. [Library Preparation]
Pellet beads on magnet until eluate is clear.
?. [Library Preparation]
Pipette off 25 μL of eluate into 1.5 mL Eppendorf LoBind DNA tube.
?. [Library Preparation]
Quantify 1 μL of eluate using Qubit HS dsDNA kit.
?. [Library Preparation]
Thaw barcodes at RT.
?. [Library Preparation]
Add 22.5 μL of end-prepped DNA, 2.5 μL Native barcode, and 25 μL of Blunt/TA ligase Master Mix.
?. [Library Preparation]
Mix gently by flicking tube and spin down.
?. [Library Preparation]
Incubate reaction mixture for 10 minutes at RT.
?. [Library Preparation]
Add 50 μL of AMPure XP beads to the reaction mixture and mix by pipetting.
?. [Library Preparation]
Incubate for 5 minutes at 55°C while occasionally flicking to mix.
?. [Library Preparation]
Spin down the sample and pellet on the magnetic rack.
?. [Library Preparation]
Pipette off supernatant, avoiding pellet.
?. [Library Preparation]
Wash beads with 200 μL of 80% EtOH. Do not disturb the pellet. Remove EtOH.
?. [Library Preparation]
Repeat step 31
?. [Library Preparation]
Spin down the tube and place the tube back in the magnetic rack.
?. [Library Preparation]
Remove any residual EtOH.
?. [Library Preparation]
Allow beads to briefly dry.
?. [Library Preparation]
Remove the tube from the magnetic rack, and resuspend in 25 μL of prewarmed 55°C nuclease-free water.
?. [Library Preparation]
Incubate for 2 minutes at 55°C.
?. [Library Preparation]
Pipette off 25 μL of eluate into 1.5 mL Eppendorf LoBind DNA tube.
?. [Library Preparation]
Quantify 1 μL of eluate using the Qubit HS dsDNA kit.
?. [Library Preparation]
Mix together 22.5 μL of end-prepped DNA, 2.5 μL of Native barcode, and 25 μL of Blunt/TA ligase Master Mix (in that order).
?. [Library Preparation]
Spin the tube down.
?. [Library Preparation]
Incubate the reaction for 10 minutes at RT.
?. [Library Preparation]
Add 50 μL of resuspended AMPure XP beads to the reaction and mix by pipetting.
?. [Library Preparation]
Incubate at 55°C for 5 minutes while occasionally flicking to mix.
?. [Library Preparation]
Spin down sample(s) and pellet on the magnetic rack.
?. [Library Preparation]
Pipette off the supernatant.
?. [Library Preparation]
Wash beads with 200 μL of 80% EtOH. Do not disturb the pellet. Remove EtOH.
?. [Library Preparation]
Repeat step 47
?. [Library Preparation]
Spin down the tube and place the tube back in the magnetic rack.
?. [Library Preparation]
Remove any residual EtOH.
?. [Library Preparation]
Allow the beads to briefly dry.
?. [Library Preparation]
Remove the tube from the magnetic rack, and resuspend in 26 μL of nuclease-free water.
?. [Library Preparation]
Incubate for 2 minutes at RT.
?. [Library Preparation]
Pellet beads on the magnet until clear and colorless.
?. [Library Preparation]
Transfer 26 μL into clear, sterile 1.5 mL Eppendorf DNA LoBind tube.
?. [Library Preparation]
Quantify 1 μL of eluate using Qubit HS dsDNA kit.
?. [Library Preparation]
Pool equimolar amounts of each barcoded sample into a 1.5 mL Eppendorf DNA LoBind tube (
?. [Library Preparation]
Quantify 1 μL of eluate using Qubit HS dsDNA kit.
?. [Library Preparation]
Dilute pooled sample to 50 μL in Nuclease-free water.
?. [Pooled Library Preparation]
Mix together 50 μL pooled barcoded samples sample, 20 μL barcode Adapter Mix (BAM), 20 μL Blunt/TA Master Mix, 10 μL Quick T4 DNA ligase.
?. [Pooled Library Preparation]
Mix by flicking the tube.
?. [Pooled Library Preparation]
Spin down the sample.
?. [Pooled Library Preparation]
Incubate the reaction for 10 minutes at RT.
?. [Pooled Library Preparation]
Add 40 μL of resuspended AMPure XP beads to the adapter ligation reaction and mix by pipetting.
?. [Pooled Library Preparation]
Incubate for 5 minutes at RT, occasionally mixing by flicking.
?. [Pooled Library Preparation]
Place on a magnetic rack and allow beads to pellet.
?. [Pooled Library Preparation]
Pipette off supernatant.
?. [Pooled Library Preparation]
Wash beads with 140 μL of ABB buffer.
?. [Pooled Library Preparation]
Resuspend beads in ABB buffer by flicking the tube.
?. [Pooled Library Preparation]
Pellet beads on the magnetic rack.
?. [Pooled Library Preparation]
Remove residual ABB buffer.
?. [Pooled Library Preparation]
Repeat step 68 - 71
?. [Pooled Library Preparation]
Remove the tube from the magnetic tube and resuspend pellet in 15 μL of Elution buffer.
?. [Pooled Library Preparation]
Incubate for 10 minutes at RT.
?. [Pooled Library Preparation]
Pellet beads on the magnetic tube rack until the eluate is clear and colorless.
?. [Pooled Library Preparation]
Mix by flicking between each sequential addition.
?. [Library Preparation]
Mix by gently flicking.
?. [DNA Fragmentation]
To achieve ~ XXX bp, spini the tube at 6000 x g.
?. [Pooled Library Preparation]
Pipet up and retain the 15 μL of eluate into a clean 1.5 mL Eppendorf DNA LoBind tube
?. [Pooled Library Preparation]
Place the tube of eluate (library) on ice until required fro library loading
?. [Pooled Library Preparation]
Use 1 μL of DNA library to Quantify using Qubit HS dsDNA assay kit.