TITLE: McGill Nanopore Ligation LibPrep Protocol SQK-LSK109
AUTHORS: Sarah Reiling, Shu-Huang Chen, Ioannis Ragoussis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to for both amplicon and high molecular weight DNA library preparations from genomic DNA using Nanopore ligation kit (SQK-LSK109). It contains no fragmentation and no PCR step. However, a fragmentation step is optional. </div></div>

[STEPS]
?. [DNA repair and end-prep]
Prepare 1.5 ug of genomic DNA into a 1.5 ml Eppendorf DNA LoBind tube. Adjust the volume to 48 uL with nuclease-free water. Mix by flicking the tube and spin down briefly.
1.5 ug genomic DNA is the default amount. We are aiming to have 5-50 fmol for MinION / PromethION loading and 3-20 fmol for Flongle loading. Depending on the fragment size, the amount of starting material may vary. If you can, aim for 10x the amount of starting material than what is needed for flow cell loading.
?. [DNA repair and end-prep]
Mix the following components in a 0.2 mL 8-strip tube; Component                                                         VolumeDNA                                                                    NEBNext FFPE DNA Repair Buffer                NEBNext FFPE DNA Repair Mix                     Ultra II End-prep reaction buffer                    Ultra II End-prep enzyme mix                         Total
48 µl
3.5 µl
2 µl
3.5 µl
3 µl
60 µl
?. [DNA repair and end-prep]
Mix gently by flicking the rube and spin down.
?. [DNA repair and end-prep]
Incubate the reaction as follows:20 °C 65 °C
?. [DNA repair and end-prep]
After the incubation, transfer the DNA to a clean 1.5 mL Eppendorf DNA LoBind tube.
?. [DNA repair and end-prep]
Resuspend the SPRI beads by vortexing.
?. [DNA repair and end-prep]
Add 60 uL of resuspended SPRI beads to the end-prep reaction an mix by flicking the tube.
?. [DNA repair and end-prep]
Incubate the sample on a rotator mixer for 5 min at room temperature.
?. [DNA repair and end-prep]
Prepare 500 uL of fresh 80% ethanol in nuclease-free water.
?. [DNA repair and end-prep]
Spin down the sample and put on a magnet for 5 min. Keep the tube on the magnet and remove the supernatant.
?. [DNA repair and end-prep]
Keep the tube on the magnet and wash the beads with 200 uL of fresh 80% ethanol without disturbing the beads for 30 seconds. Remove the ethanol and discard. Repeat the previous wash step.
?. [DNA repair and end-prep]
Quick spin the tube and place it back on the magnet rack. Pipette off the residual ethanol. Air-dry for 30 seconds, do not over dry the pellet.
?. [DNA repair and end-prep]
Remove the tube from the magnet and resuspend the beads in 61 uL of nuclease-free water. Incubate for 10 min at 37 °C.
?. [DNA repair and end-prep]
Quick spin the tube and place it on a magnet rack for 5 min.
?. [DNA repair and end-prep]
Transfer 61 uL of elute DNA into a clean 1.5 mL Eppendorf DNA LoBind tube.
?. [DNA repair and end-prep]
Quantify 1 uL of eluted DNA using a fluorimetric dsDNA assay.
Continue the end-repaired DNA into the adapter ligation step. It is also possible to store the sample at 4°C overnight at this point.
?. [Adapter ligation step]
Thaw Ligation buffer (LNB) and Elution buffer (EB) at room temperature, spin down and mix by pipetting. Place on ice immediately after thawing and mixing.
Due to viscosity, vortexing the LNB buffer is not effective, pipetting the buffer to mix well.
?. [Adapter ligation step]
Spin down the Adapter Mix (AMX) and Quick T4 Ligase, and place on ice.
?. [Adapter ligation step]
Thaw one tube of Long Fragment buffer (LFB) or Short Fragment buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.
Wash buffer LFB and SFB are designer to enrich DNA and purify all fragments equally after adapter ligation. LFB is used to enrich long DNA fragments of >3 kb and SFB is to retain DNA fragments of all size.
?. [Adapter ligation step]
In a 1.5 mL Eppendorf DNA LoBind tube, mix the following order:Component                                                                      VolumeEnd-repaired DNA from the previous step               Ligation Buffer (LNB)                                                  NEBNext Quick T4 DNA Ligase                                 Adapter Mix (AMX)                                                      Total
60 µl
25 µl
10 µl
5 µl
100 µl
?. [Adapter ligation step]
Mix gently by flicking the tube and spin down.
?. [Adapter ligation step]
Incubate the reaction for 10 minutes at room temperature.
?. [adapter ligated DNA purification]
Resuspend the SPRI beads by vortexing.
?. [adapter ligated DNA purification]
Incubate the tube on a rotator mixer for 5 minutes at room temperature.
?. [adapter ligated DNA purification]
Add 50 µL of resuspended SPRI beads to the ligation reaction and mix by flicking the tube.
?. [adapter ligated DNA purification]
Spin down the sample and put on a magnet for 5 min. Keep the tube on the magnet and remove the supernatant.
?. [adapter ligated DNA purification]
Remove the tube from the magnet and wash the beads with 250 uL of Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB). Flick gently to resuspend the beads, leave for 5 min at room temperature, then return the tube to the magnetic rack and let the beads to rebind. Remove the supernatant and discard.
?. [adapter ligated DNA purification]
Repeat the previous wash step with LFB or SFB.
?. [adapter ligated DNA purification]
Quick spin the tube and place it back on the magnet rack. Pipette off the residual ethanol. Air-dry for 30 seconds, do not over dry the pellet.
?. [adapter ligated DNA purification]
Remove the tube from the magnet and resuspend the beads in 25 uL of elution buffer (EB) for MinION and PromethION libraries and 15 ul for Flongle libraries. Incubate for 30 min at 37 °C to increase the recovery of long fragment DNA.