TITLE: ONT dA-tailing for Fungal Barcoding
AUTHORS: Stephen       Douglas Russell

[DESCRIPTION]
This protocol is for dA-tailing, which is an enzymatic method for adding a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA molecule. In other words, this puts A-chains on the end of our PCR product, creating a site for the ligation adapter to attach to. Simple process - create a reaction with three chemicals, cleanup the product with beads.

Time required: ~45 minutes

The NEB protocol this is based on can be found here.

[STEPS]
SECTION: End repair/A-tailing
1. Put a 1.5mL tube of molecular water on a heat block at 55 °C. This will be used after the cleanup step towards the end of this protocol.

Thaw the NEB End-prep Reaction Buffer and NEB End-prep Enzyme Mix at room temperature. (Won't take long.)

Also if you are continuing on with adapter ligation (next protocol) once this step is done, it would be good to set the chems for next step on ice so they can begin to thaw as well. 

This includes LA, Quick T4 Ligase, LNB, EB, and SFB. (if using V14 chemistry)

SECTION: End repair/A-tailing
2. Optional: Thaw DNA CS at room temperature, spin down for 5 s, mix by pipetting, and place on ice. 

DNA CS -   

(DNA CS consists of a standard DNA sequence that can be used to provide quality control for sequencing and alignment. Generally unnecessary for DNA barcoding efforts with hundreds of repeats of a single amplicon. I usually skip it)
SECTION: End repair/A-tailing
3. Mix your amplicon DNA pool thoroughly with a pipette (pipette up and down 5 times). Briefly spin down for 5 s.

SECTION: End repair/A-tailing
4. Important: Vortex the Ultra II End-prep Reaction Buffer for 30 seconds.

(Do not vortex the End-prep Enzyme Mix)
SECTION: End repair/A-tailing
5. In a 0.2mL thin wall, sterile, nuclease-free tube, combine the following in order. Mix each reagent together after it is added by gently pipetting the entire volume up and down 10-20 times for each addition.

Ideal amplicon DNA concentration is 0.5ng per 50mL for Flongle or 1ug DNA per 50uL for R10.4.1. At this concentration you can use the volumes described below.

Component                                            R10.4.1 Flongle Volume        R10.4.1 MinION Flowcell Volume
DNA CS (optional)                                 0.5uL                                        1uL
Amplicon DNA                                       24.5uL (0.5ng)                         49uL (1ng)
Ultra II End-prep reaction buffer          3.5uL                                        7uL
Ultra II end-prep enzyme mix               1.5uL                                        3uL
Total                                                        30uL                                        60uL

The NEB protocol this is based on can be found here.
SECTION: End repair/A-tailing
6. Spin down the tube in a mini centrifuge for 5 s. 

SECTION: End repair/A-tailing
7. Incubate in a thermocycler using the following program:

20 °C for 5 minutes
65 °C for 5 minutes
4 °C Hold
SECTION: End repair/A-tailing
8. Spin down the tube for 5 seconds in a mini centrifuge.

SECTION: End repair/A-tailing
9. Transfer the entire 30 µL / 60 µL reaction to a new 1.5mL LoBind eppi tube.

SECTION: End repair/A-tailing
10. Resuspend magnetic beads in solution by vortexing. Add 30 µL (Flongle) - 60 µL (MinION) of beads to the reaction (1X bead cleanup) and mix gently by pipetting up and down.

SECTION: End repair/A-tailing
11. Incubate at room temperature for 5 min. (Can put the tube in a rotator [hula] mixer if one is available. I typically do not use one; just let it sit in a tube rack.)

SECTION: End repair/A-tailing
12. Spin down the tube in a mini centrifuge for 5 -10 seconds.


SECTION: End repair/A-tailing
13. Place sample tube on the magnetic separator for 2 min or until the solution clears. Beads should now be on the side of the tube.
SECTION: End repair/A-tailing
14. With the tube still on the magnet, remove the liquid from the tube and discard. Be sure not to disturb the beads.
SECTION: End repair/A-tailing
15. With the tube still on the magnet, add 200 µL of 80% ethanol to the tube and let sit for 2 min. Try to minimize disturbance of the beads. Fill gently with liquid stream from the pipette tip on opposite side of the beads.

SECTION: End repair/A-tailing
16. Remove ethanol by pipetting and discard.
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17.  Repeat the ethanol wash one time.   

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18. Spin down for 5 s and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.

SECTION: End repair/A-tailing
19. Remove the tube from the magnet and add 31 µL for Flongle or 61 µL of molecular water for MinION. Pipette up and down five times to mix until the pellet is fully suspended. 

The DNA will now be released from the beads and suspended in the water.

SECTION: End repair/A-tailing
20. Incubate for 2 min at room temperature.
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21. Place the tube back on the magnet for 2 min or until the solution is clear.

SECTION: End repair/A-tailing
22. Transfer the water containing the DNA to a new 1.5mL LoBind eppi tube.

You should now have your A-tailed DNA template.
SECTION: DNA Quantification
23. If you have access to a Quantus or Qubit fluorometer, now is a good time to quantify the resulting amount of DNA in your purified sample. 

If not, the 31 µL / 61 µL of molecular water added above should put you in the ballpark of the right DNA concentration.

I typically have a concentration of 11-33 ng/uL at this point and used it at this level for the next step. No dilutions or adjustments.

SECTION: DNA Quantification
24. It is possible to break and store the sample at 4C overnight if needed. It would be ideal to continue on to adapter ligation at this time.