3RAD Lab Protocol

Modified from the Oaks Lab protocol (https://github.com/phyletica/lab-protocols/blob/master/3rad-protocol.md) and the BadDNA @ Univ. Georgia protocol (https://baddna.uga.edu/protocols.html)

0. Quantify and Normalize Samples

Before beginning the 3RAD protocol, you will first quantify the DNA concentration of each sample (we perform this step using a Qubit fluoremeter).

Following quantification, you will then standardize your samples to 20 ng/µL.

1. Digestion

Note: Step 2 (Ligation) must occur immediately following Step 1.

Materials Needed:

• 20,000 U/mL New England BioLabs (NEB) Restriction Enzymes
  • XbaI
  • EcoRI-HF
  • NheI-HF
• molecular grade H2O
• 10X NEB CutSmart Buffer
• 2.5 µM Read 1 (i5) and Read2 (i7) Adapters
  • a working stock will need to be made before beginning. Our lab creates several i5 adapter sets in 8-strip PCR tubes and i7 adapter sets in a 12-strip for easy grab use. DO NOT THAW UNUSED STOCK.
• Standardized DNA (20 ng/µL)

Materials Note: only take out enzymes/polymerase from freezer when they are ready to be used. No need to thaw unless specifically mentioned below!

1a. Create Master Mix and Add Adapters + DNA

Master Mix

Enzyme Digestion	1X	106X
NEB CutSmart	1.5 µL	159 µL
dH2O	3.0 µL	318 µL
XbaI	0.5 µL	53 µL
EcoRI-HF	0.5 µL	53 µL
NheI-HF	0.5 µL	53 µL
Total Volume	6 µL	642 µL

vortex master mix upon completion

1. Take a strip of eight 0.5 mL PCR tubes and pipette 78 µL into each tube to use as aliquot for use of multi-channel pipetting.

2. Place 6 µL of master mix in each well using a multi-channel pipette.

3. Place 2 µL of each adapter in correct well using a multi-channel pipette (see image below for plate orientation).

• use 12-channel pipette for i7 adapters.
• use 8-channel pipette for i5 adapters.

4. Place 5 µL of DNA in each well after adding adapters using a multi-channel pipette.

5. Seal 96-well plate with adhesive film and proceed to 1b.

1b. Incubate

1. Incubate samples at 37°C for 1 hour with no heated lid.
2. Spin down plate after incubation.
3. Place on ice block before proceeding with Step 2.

2. Ligation

Materials Needed:

• molecular grade H20
• Promega 10mM rATP
  • note: need to thaw (hold in hand for last 3-5 minutes of ibcubation in step 1)
• 10X Ligase Buffer
  • note: heat to dissolve with heat block if not in solution. Preferably start this before Step 1
• 400,000 U/mL NEB T4 DNA Ligase

2a. Create Master Mix and Add

Master Mix

Ligation Mix	1X	106X
dH2O	2.75 µL	291.5 µL
rATP	1.5 µL	159 µL
Ligase Buffer	0.5 µL	53 µL
DNA Ligase	0.25 µL	26.5 µL
Total Volume	5 µL	530 µL

vortex master mix upon completion

1. Take a strip of eight 0.5 mL PCR tubes and pipette 65 µL into each tube to use as aliquot for use of multi-channel pipetting.
2. Using a multi-channel pipette, add 5 µL of the master mix to each well.
3. Seal 96-well plate with adhesive film and proceed to 2b.

2b. Incubation

1. Incubate samples without heated lid

• 2 cycles of:
  • 22 °C for 20:00
  • 37 °C for 10:00
• 80 °C for 20:00
  • reduce to 10 °C

2. Spin down plate after incubation.

Note: While the samples incubate, make fresh 70% EtoH for Bead Cleanup

3. Bead Cleanup #1

Materials Needed:

• molecular grade H20
• Omega BioTek Beads
• 70% EtOH
• TLE Buffer

1. Spin down PCR product after removing from thermal cycler.
2. Pool 5 µL of each ligation product into 1.5 mL tube.
3. Mix pooled ligation product with 1X beads and vortex + spin down.
   • Sit for 5 minutes.
   • Place on magnet and wait until clear.
   • Carefully remove and discard the supernatent without disturbing the beads.
4. Wash with 70% EtOH.
   • Add 500 µL 70% EtOH and let sit for 2 minutes on magnet.
   • Pipette EtOH from tube.
   • Repeat, then spin down.
   • Get drops of EtOH with 10 µL pipette tip and let sit for 2 minutes with lid open until EtOH is gone.
5. Repeat Step 4.
6. Resuspend in 25 µL TLE Buffer.
   • Remove from magnet, flick bead down, vortex + spin down.
   • Let sit for 5 minutes.
   • Place back on magnet until clear (~ 3 minutes).
   • Transfer cleaned ligation product to new tube.

4. One Cycle PCR with iTru5-8N Primer

Materials Needed:

• molecular grade H20
• 5X Kappa HiFi Buffer
• 10µM dNTP
• Kappa HiFi Polymerase (HotStart)
• 5 µM iTru5 Primer
  • need to make 5 µM primer beforehand (starts at 100 µM)

Note: At this point, I take all of the materials out and place into a cooling block in the fridge except for the HotStart for easy assess.

4a. Create Master Mix and Add DNA

Master Mix

PCR Mix	1X	5X
dH2O	29.5 µL	147.5 µL
Kappa Buffer	10 µL	50 µL
iTru5 Primer	3 µL	15 µL
dNTP	1.5 µL	7.5 µL
Kappa HiFi Polymerase	1.0	5.0
Total Volume	45 µL	225 µL

vortex master mix upon completion

1. Add 45 µL of master mix to four PCR tubes.
2. Add 5 µL pooled DNA to each PCR tube.
3. Spin down tubes.

4b. Incubation

1. Incubate samples with heated lid

• 95 °C for 2:00
• 60 °C for 0:30
• 72 °C for 5:00

2. Spin down PCR product after removing from thermal cycler.
3. Pool PCR products in one tube.

5. Bead Cleanup #2

1. Repeat steps for Bead Cleanup #1 but use 1.5X beads

6. Two Primer Amplification with P5 and iTru7 Primers

Materials Needed:

• molecular grade H20
• 5X Kappa HiFi Buffer
• 5 µM iTru7 Primer
  • need to make 5 µM primer beforehand (starts at 100 µM)
  • see note in step 6a about iTru7 Primer(s)
• 5 µM P5 Primer
  • need to make 5 µM primer beforehand (starts at 100 µM)
• 10µM dNTP
• Kappa HiFi Polymerase (HotStart)

6a. Create Master Mix and Add DNA

Master Mix

PCR Mix	1X	5X
dH2O	26.5 µL	132.5 µL
Kappa Buffer	10 µL	50 µL
P5 Primer	3 µL	15 µL
dNTP	1.5	7.5
Kappa HiFi Polymerase	1.0	5.0
Total Volume	42 µL	210 µL

vortex master mix upon completion

1. Add 42 µL into four PCR tubes.
2. Add 3 µL of one iTru7 Primer to one tube (repeat four times - see below).
   • For the iTru7 Primer, you will add one indexed primer to one tube, respectively. I.e., i7 101-1 will go into tube 1, i7 101-2 will go into tube 2, etc.
   • **Make sure the primer combination you decide to use are suitable for multiplexing on a NovaSeq.
3. Add 5 µL pooled 8N PCR product to each tube, then spin down.

6b. Incubation

1. Incubate samples with heated lid

• 95 °C for 3:00
• 6 cycles of:
  • 98 °C for 0:20
  • 60 °C for 0:15
  • 72 °C for 0:30
• 72 °C for 5:00

2. Spin down PCR product after removing from thermal cycler.
3. Pool PCR products in one tube.

7. Bead Cleanup #3

1. Repeat steps for Bead Cleanup #1 but use 1.5X beads

• resuspend in 35µL of TLE Buffer

8. Size selection

1. Size select DNA using BluePippin

Note: blue pippen cassettes can be stored for re-use of unused lanes

9. Final Amplification with P5 and P7 Primers

Materials Needed:

• molecular grade H20
• 5X Kappa HiFi Buffer
• 5 µM P5 Primer
• 5 µM P7 Primer
  • need to make 5 µM primer beforehand (starts at 100 µM)
• 10µM dNTP
• Kappa HiFi Polymerase (HotStart)

9a. Create Master Mix and Add DNA

Master Mix

PCR Mix	1X	5X
dH2O	26.5 µL	132.5 µL
Kappa Buffer	10 µL	50 µL
P5 Primer	3 µL	15 µL
P7 Primer	3 µL	15 µL
dNTP	1.5	7.5
Kappa HiFi Polymerase	1.0	5.0
Total Volume	45 µL	225 µL

vortex master mix upon completion

1. Add 45 µL into four PCR tubes.
2. Add 5 µL pooled 8N PCR product to each tube, then spin down.

9b. Incubation

1. Incubate samples with heated lid

• 95 °C for 3:00
• 6 cycles of:
  • 98 °C for 0:20
  • 60 °C for 0:15
  • 72 °C for 0:45
• 72 °C for 5:00

2. Spin down PCR product after removing from thermal cycler.
3. Pool PCR products in one tube.

10. Bead Cleanup #4

1. Repeat steps for Bead Cleanup #1 but use 1X beads
2. Re-suspend in 25 µL 10 µM TrisHCL buffer.