TITLE: BestRAD protocol
AUTHORS: Thom Nelson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Modified from protocol of Sean O’Rourke and Mike Miller published in:</div></div>

[STEPS]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
I. Restriction DigestDilute genomic DNA samples to a common concentration.Add  (50-100 ng) of genomic DNA to each well of a 96-well PCR plate.
10 µl
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Please select between the two following options: performing sheared, single-digest RADperforming double-digest
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Into 1.5 ml tube, make restriction digest master mix (vols for 100 rxns).76.2 μL water 134.4 μL 10X NEBuffer 3.1 13.4 μL PstI (NEB R0140L)
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[water ]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[10X NEBuffer 3.1 ]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[PstI (NEB R0140L)]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Pipette equal volumes of master mix into each tube of an 8-tube strip.
~  ea for full 96-well plate.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
To each 96 plate well, pipet .
[PstI digestion master mix]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
In a thermal cycler: a)  for . b)  for . c) Slow ramp to  — /sec
37 °C
80 °C
4 °C
0.1 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Perform first digestion: BfaI master mix (add components to a 1.5 mL tube)For full 96-well plate: For 1/2 plate (48):
For optimal enzyme activity and minimal star activity, BfaI and PstI need to be used sequentially. PstI has lower activity and star activity in low-salt buffers like CutSmart, while BfaI has very reduced activity in high-salt buffers like NEB 3.1. Other enzyme combinations may be combined into a single digest step.
[water]
[10X CutSmart Buffer ]
[BfaI (NEB R0568S)]
[water ]
[10X CutSmart Buffer ]
[BfaI]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
For full 96-well plate: Add .For 1/2 plate (48): Add .
[water]
[water ]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
For full 96-well plate: Add . For 1/2 plate (48): Add .
[10X CutSmart Buffer ]
[BfaI (NEB R0568S)]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
For full 96-well plate: Add .For 1/2 plate (48): Add .
[10X CutSmart Buffer ]
[BfaI]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Pipette equal volumes of master mix into each tube of an 8-tube strip.
~  ea for full 96-well plate.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Pipette  into each well.
[BfaI master mix]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Vortex plate to mix.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Quick spin to collect liquid.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
In a thermal cycler:  for .  for . Slow ramp to  — /sec
37 °C
80 °C
4 °C
0.1 °C
On Fishman Lab thermal cyclers, program available at THOM>CUTSMART.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Perform second digestion:PstI master mix (add components to a 1.5 mL tube) For a full 96-well plate 51.5 μL water 156.8 μL NEBuffer 3.1 15.7 μL PstI (NEB R0140L) For 48 reactions 25.8 μL water 78.4 μL NEBuffer 3.1 7.8 μL PstIFor 48 reactions 25.8 μL water 78.4 μL NEBuffer 3.1 7.8 μL PstI
?. [Part 1 - Digestion and BestRAD adaptor ligation]
For a full 96-well plate: Add .For 48 reactions: Add .
[water]
[water]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
For a full 96-well plate: Add .For 48 reactions: Add .
[NEBuffer 3.1]
[NEBuffer 3.1]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
For a full 96-well plate: Add .For 48 reactions: Add .
[PstI (NEB R0140L)]
[PstI]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Pipette equal volumes of master mix into each tube of an 8-tube strip.
~  ea for full 96-well plate.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Pipette  into each well.
[PstI max mix]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Vortex plate and quick spin.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
In a thermal cycler:  for .   for . Slow ramp to  — /sec
37 °C
80 °C
4 °C
0.1 °C
On Fishman Lab thermal cyclers, program available at THOM>CUTSMART.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
II. Adaptor LigationAdd .
This part assumes   ligation volume and and use of NEB3.1 in the previous step. If only CutSmart buffer was used for digestion, supplement ligation buffer with fullstrength NEB2.1 (  per well).
[annealed BestRad SbfI/PstI adaptors (50 nM)]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Make ligation master mix (vols for 100 rxns):
[water ]
[NEB2.1 ]
[rATP (100 mM, Fermentas R0441) ]
[Ligase (NEB M0202L)]
From S. Bassham: NEB Buffer 2 is used in the ligation reactions in this protocol instead of ligase buffer because the salt it contains (50 mM NaCl) ensures the double-stranded adapters remain annealed during the reactions. T4 DNA Ligase is active in all 4 NEB Buffers if supplemented with 1mM rATP, but doesn’t work at maximum efficiency in NEB 3.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[water ]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[NEB2.1 ]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[rATP (100 mM, Fermentas R0441) ]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[Ligase (NEB M0202L)]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Pipette  into each tube of an 8-tube strip for multichannel distribution.
[master mix]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Into each 96 plate well, pipet .
[ligation master mix]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Incubate plate at  overnight.
20 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
III. NEXT DAYHeat kill the ligation Kill the ligation reactions by deactivating ligase at .In thermal cycler, incubate plate at  for .
65 °C
65 °C
On Fishman Lab thermal cyclers, program available at THOM>LIGKILL.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Kill the ligation reactions by deactivating ligase at .
65 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
In thermal cycler, incubate plate at  for .
65 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Multiplex 48 barcoded samples 1. Using an 8-channel pipettor:     a) transfer  of each adaptor-ligated sample into an 8-tube PCR strip.2. Combine pooled samples into a single 1.5 mL tube. 3. Split the multiplex into two or more aliquots.     a) The following steps assume . 4. Store plate at  for future multiplexing.
10 µl
[multiplexed DNA]
-20 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Using an 8-channel pipettor, transfer  of each adaptor-ligated sample into an 8-tube PCR strip.
10 µl
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Combine pooled samples into a single 1.5 mL tube.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Split the multiplex into two or more aliquots.
The following steps assume  .
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Store plate at  for future multiplexing.
-20 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
II. Adaptor LigationAdd .
This part assumes   ligation volume and and use of NEB3.1 in the previous step. If only CutSmart buffer was used for digestion, supplement ligation buffer with fullstrength NEB2.1 (  per well).
[annealed BestRad SbfI/PstI adaptors (50 nM)]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Make ligation master mix (vols for 100 rxns):
[water ]
[NEB2.1 ]
[rATP (100 mM, Fermentas R0441) ]
[Ligase (NEB M0202L)]
From S. Bassham: NEB Buffer 2 is used in the ligation reactions in this protocol instead of ligase buffer because the salt it contains (50 mM NaCl) ensures the double-stranded adapters remain annealed during the reactions. T4 DNA Ligase is active in all 4 NEB Buffers if supplemented with 1mM rATP, but doesn’t work at maximum efficiency in NEB 3.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[water ]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[NEB2.1 ]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[rATP (100 mM, Fermentas R0441) ]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Add .
[Ligase (NEB M0202L)]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Pipette  into each tube of an 8-tube strip for multichannel distribution.
[master mix]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Into each 96 plate well, pipet .
[ligation master mix]
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Incubate plate at  overnight.
20 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
III. NEXT DAYHeat kill the ligation Kill the ligation reactions by deactivating ligase at .In thermal cycler, incubate plate at  for .
65 °C
65 °C
On Fishman Lab thermal cyclers, program available at THOM>LIGKILL.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Kill the ligation reactions by deactivating ligase at .
65 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
In thermal cycler, incubate plate at  for .
65 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Multiplex 48 barcoded samples 1. Using an 8-channel pipettor:     a) transfer  of each adaptor-ligated sample into an 8-tube PCR strip.2. Combine pooled samples into a single 1.5 mL tube. 3. Split the multiplex into two or more aliquots.     a) The following steps assume . 4. Store plate at  for future multiplexing.
10 µl
[multiplexed DNA]
-20 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Using an 8-channel pipettor, transfer  of each adaptor-ligated sample into an 8-tube PCR strip.
10 µl
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Combine pooled samples into a single 1.5 mL tube.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Split the multiplex into two or more aliquots.
The following steps assume  .
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Store plate at  for future multiplexing.
-20 °C
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Concentrate sample to 210 μL with Ampure 1X. Divide sample into two Bioruptor tubes.
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Run  on the fragment analyzer NGS mode to assay shearing efficiency.
2 µl
?. [Part 1 - Digestion and BestRAD adaptor ligation]
Sonicate with BioRuptor NGS: 9 cycles: ,
[on]
[off]
?. [Part 2 - RAD-tag isolation]
I. Before beginning the following steps, set a wet or dry bath to .
56 °C
?. [Part 2 - RAD-tag isolation]
II. Prepare Dynabead M280 streptavidin magnetic beads.Adjust bead volume.
?. [Part 2 - RAD-tag isolation]
2X Binding and Wash (B+W) Buffer.
[TrisHCl (pH 7.5) ]
[EDTA pH 8.0 ]
[NaCl]
?. [Part 2 - RAD-tag isolation]
Transfer  to a new 1.7 ml tube.
[Dynabeads]
?. [Part 2 - RAD-tag isolation]
Place tube in magnetic rack and remove supernatant.
?. [Part 2 - RAD-tag isolation]
Wash the beads (1/2) Mix Quick spin Remove supernate
[2X B+W buffer ]
?. [Part 2 - RAD-tag isolation]
Add .
[2X B+W buffer ]
?. [Part 2 - RAD-tag isolation]
Mix for .
?. [Part 2 - RAD-tag isolation]
Quick spin.
?. [Part 2 - RAD-tag isolation]
Remove supernate.
?. [Part 2 - RAD-tag isolation]
Wash the beads (2/2) Mix Quick spin Remove supernate
[2X B+W buffer ]
?. [Part 2 - RAD-tag isolation]
Add .
[2X B+W buffer ]
?. [Part 2 - RAD-tag isolation]
Mix for .
?. [Part 2 - RAD-tag isolation]
Quick spin.
?. [Part 2 - RAD-tag isolation]
Remove supernate.
?. [Part 2 - RAD-tag isolation]
Resuspend beads in X μL 2X B+W buffer (X = multiplex volume).
?. [Part 2 - RAD-tag isolation]
III. Bead BindingAdd resuspended beads to ~.
[multiplexed DNA]
?. [Part 2 - RAD-tag isolation]
While DNA fragments are binding, make a dilution BW buffer 2-fold in 10 mM Tris.1. Will need ~  per multiplex 2. Split dilution into 2x 1.5mL tubes and set one tube in a  wet or dry bath.
1 ml
56 °C
?. [Part 2 - RAD-tag isolation]
Incubate at  for . Mix every  by inverting.
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Quick spinRemove liquid from cap of tube Not enough to pellet beads
?. [Part 2 - RAD-tag isolation]
Place tube on magnetic rack - Wash (1/4)Resuspend beads in .
[1X B+W Buffer]
?. [Part 2 - RAD-tag isolation]
Place tube on magnetic rack - Wash (2/4)Resuspend beads in .
[1X B+W Buffer]
?. [Part 2 - RAD-tag isolation]
Place tube on magnetic rack - Wash (3/4)Resuspend beads in .
56 °C
[1X B+W Buffer]
?. [Part 2 - RAD-tag isolation]
Place tube on magnetic rack - Wash (4/4)
Resuspend beads in .
56 °C
[1X B+W Buffer]
?. [Part 2 - RAD-tag isolation]
IV. Liberate DNA from beadsDilute an aliquot of appropriate NEBuffer 10-fold to 1X.
?. [Part 2 - RAD-tag isolation]
Resuspend beads in  (NEBuffer 3.1 for PstI).
[1X restriction digest buffer]
?. [Part 2 - RAD-tag isolation]
Place on magnetic rack, remove supernate.
?. [Part 2 - RAD-tag isolation]
Resuspend beads in .
[1X digest buffer]
?. [Part 2 - RAD-tag isolation]
Transfer to PCR tube.
?. [Part 2 - RAD-tag isolation]
Add .
[PstI]
?. [Part 2 - RAD-tag isolation]
Incubate tube at  for .
37 °C
On Fishman Lab thermal cyclers, program available at THOM>CUTSMART.
Because the next step removes the enzyme, there is no need to heat inactivate.
?. [Part 2 - RAD-tag isolation]
Quick spin.
?. [Part 2 - RAD-tag isolation]
Place tube on magnetic rack, KEEP SUPERNATE!!
?. [Part 2 - RAD-tag isolation]
V. Bead cleanup of digestion reactionAllow an aliquot of Ampure XP beads to warm to  for ~.
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Vortex prior to addition to resuspend.
?. [Part 2 - RAD-tag isolation]
Add  to ligation reaction.1. Mix very well by pipetting     a) slowly     b) expel final volume slowly due to viscosity 2. Allow to incubate at  for .
[Ampure XP beads]
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Add  to ligation reaction.
[Ampure XP beads]
?. [Part 2 - RAD-tag isolation]
Mix very well by pipetting. slowly expel final volume slowly due to viscosity
?. [Part 2 - RAD-tag isolation]
Allow to incubate at  for .
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Make fresh 80% ethanol. ≥ 1 mL per reaction.
Use aliquoted ethanol in 50 ml conical tubes to limit evaporation/hydration of ethanol.
?. [Part 2 - RAD-tag isolation]
Place on magnetic rack and allow beads to pellet.
?. [Part 2 - RAD-tag isolation]
Slowly remove supernate.
?. [Part 2 - RAD-tag isolation]
Ethanol wash (1/2) Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 2 - RAD-tag isolation]
Add .
[80% ethanol ]
?. [Part 2 - RAD-tag isolation]
Incubate for  with beads still on magnet.
?. [Part 2 - RAD-tag isolation]
Ethanol wash (2/2) Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 2 - RAD-tag isolation]
Add .
[80% ethanol ]
?. [Part 2 - RAD-tag isolation]
Incubate for  with beads still on magnet.
?. [Part 2 - RAD-tag isolation]
Remove all ethanol and allow beads to dry ~  with the lid open.
?. [Part 2 - RAD-tag isolation]
Elute DNA 1. Add  to bead pellet. 2. Wash buffer over beads and pipette to resuspend.         a) Suspension will turn a pale, even brown. 3. Incubate at  for . 4. Place tube on magnet to separate beads.5. Pipette supernate and place in a clean PCR tube.
[10 mM Tris-HCl]
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Add  to bead pellet.
[10 mM Tris-HCl]
?. [Part 2 - RAD-tag isolation]
Wash buffer over beads and pipette to resuspend.
Suspension will turn a pale, even brown.
?. [Part 2 - RAD-tag isolation]
Incubate at  for .
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Place tube on magnet to separate beads.
?. [Part 2 - RAD-tag isolation]
Pipette supernate and place in a clean PCR tube.
?. [Part 2 - RAD-tag isolation]
VI. Now is an okay time to stop if need be. Store eluted DNA at  overnight.
-20 °C
?. [Part 3 - NextGen library prep]
I. End-repair and A-tailingTo the liberated DNA, add: 1. 2.
Use full reactions NEBNext Ultra DNA Library Prep Kit for Illumina (NEB E7370S/L) with no modifications except A. Use 1:10 diluted adaptor. B. Especially if performing double-digest, use a molecular barcoded i5 adaptor (see Materials)
This step blunts single-stranded DNA ends and adds a single A overhang.
[NEBNext Ultra II End Prep Enzyme Mix ]
[NEBNext Ultra II End Prep Reaction Buffer]
?. [Part 3 - NextGen library prep]
Add  to the liberated DNA.
[NEBNext Ultra II End Prep Enzyme Mix ]
?. [Part 3 - NextGen library prep]
. Mix by pipetting 10x w/ a pipette set to 50 µl.
?. [Part 3 - NextGen library prep]
Add  to the liberated DNA.
[NEBNext Ultra II End Prep Reaction Buffer]
?. [Part 3 - NextGen library prep]
In a thermal cycler:  at  at Hold at
20 °C
65 °C
4 °C
On Fishman Lab thermal cyclers, use THOM>NEB_EP.
?. [Part 3 - NextGen library prep]
II. Adaptor ligationMake sure Ligation Master Mix is well-mixed prior to addition.
1. This part adds universal Illumina adaptors onto all end-prepped fragments. NEB NEXT adaptors form hairpins and contain a single uracil in the loop. The USER enzyme mix has endonuclease activity that cleaves the adaptor at the U, creating a Y-shaped adaptor for subsequent addition of oligos and amplification by PCR. 2. From NEB: “If input DNA ≤ 100 ng, use 1:10 diluted adaptor (diluted in 10 mM Tris, 10 mM NaCl)”. Efficient RAD preps will isolate ~1%-15% of genomic DNA. Good luck getting anything greater than 100 ng total from 48 samples!
?. [Part 3 - NextGen library prep]
Vortex quickly.
?. [Part 3 - NextGen library prep]
Quick spin.
?. [Part 3 - NextGen library prep]
To end-prepped sample, add: 1. 2. 3.
[NEBNext Ultra II Ligation Master Mix]
[NEBNext Ligation Enhancer]
[NEBNext Adaptor for Illumina (diluted 1:10 from stock)]
?. [Part 3 - NextGen library prep]
Add .
[NEBNext Ultra II Ligation Master Mix]
?. [Part 3 - NextGen library prep]
Add .
[NEBNext Ligation Enhancer]
?. [Part 3 - NextGen library prep]
Add .
[NEBNext Adaptor for Illumina (diluted 1:10 from stock)]
?. [Part 3 - NextGen library prep]
Mix sample by pipetting 10X with a pipette set to 80 µl.
?. [Part 3 - NextGen library prep]
Quick spin to collect any liquid from the side of the tube.
?. [Part 3 - NextGen library prep]
In a thermal cycler: 1. Either a) keep the thermal cycler lid open OR b) manually turn off heated lid 2.  at
20 °C
On Fishman Lab thermal cyclers, use THOM>NEB_LIG.
?. [Part 3 - NextGen library prep]
Add  to the ligation mixture.
[USER enzyme]
?. [Part 3 - NextGen library prep]
Mix sample by pipetting 10X with a pipette set to 80 µl.
?. [Part 3 - NextGen library prep]
In a thermal cycler with heated lid: 1.  at
37 °C
On Fishman Lab thermal cyclers, use THOM>NEBUSER.
?. [Part 3 - NextGen library prep]
III. Bead cleanup of ligation reactionAllow an aliquot of Ampure XP beads to warm to  for ~.
0 Room temperature
?. [Part 3 - NextGen library prep]
Vortex prior to addition to resuspend.
?. [Part 3 - NextGen library prep]
Make fresh 80% ethanol. ≥ 1 mL per reaction.
?. [Part 3 - NextGen library prep]
Add  to ligation reaction.1. Mix very well by pipetting.        a) slowly         b) expel final volume slowly due to viscosity 2. Allow to incubate at  for .
[Ampure XP beads]
0 Room temperature
?. [Part 3 - NextGen library prep]
Add  to ligation reaction.
[Ampure XP beads]
?. [Part 3 - NextGen library prep]
Mix very well by pipetting.        a) slowly         b) expel final volume slowly due to viscosity
?. [Part 3 - NextGen library prep]
Allow to incubate at  for .
0 Room temperature
?. [Part 3 - NextGen library prep]
Make fresh 80% ethanol. ≥ 1 mL per reaction.
?. [Part 3 - NextGen library prep]
Place on magnetic rack and allow beads to pellet.
?. [Part 3 - NextGen library prep]
Slowly remove supernate.
?. [Part 3 - NextGen library prep]
Ethanol wash (1/2)Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Add .
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Incubate for  with beads still on magnet.
?. [Part 3 - NextGen library prep]
Ethanol wash (2/2)Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Add .
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Incubate for  with beads still on magnet.
?. [Part 3 - NextGen library prep]
Remove all ethanol and allow beads to dry ~  with the lid open.
?. [Part 3 - NextGen library prep]
Elute DNA Add  to bead pellet.Wash buffer over beads and pipette to resuspend.Incubate at    for  .Place tube on magnet to separate beads. Pipette supernate and place in a a clean PCR tube.Incubate at   for .Place tube on magnet to separate beads. Pipette supernate and place in a a clean PCR tube.
[10 mM Tris-HCl]
0 Room temperature
?. [Part 3 - NextGen library prep]
Add  to bead pellet.
[10 mM Tris-HCl]
?. [Part 3 - NextGen library prep]
Wash buffer over beads and pipette to resuspend.
?. [Part 3 - NextGen library prep]
Incubate at   for .
0 Room temperature
?. [Part 3 - NextGen library prep]
Place tube on magnet to separate beads.
?. [Part 3 - NextGen library prep]
Pipette supernate and place in a a clean PCR tube.
?. [Part 3 - NextGen library prep]
IV. PCR enrichment of adaptor-ligated fragmentsTo the purified DNA, add:   — USE i5 WITH MOLECULAR BARCODE
[NEBNext Ultra II Q5 Master Mix]
[i7 index primer ]
[i5 universal primer]
?. [Part 3 - NextGen library prep]
Add  to the purified DNA.
[NEBNext Ultra II Q5 Master Mix]
?. [Part 3 - NextGen library prep]
Add  to the purified DNA.
[i7 index primer ]
?. [Part 3 - NextGen library prep]
Add  to the purified DNA. — USE i5 WITH MOLECULAR BARCODE
[i5 universal primer]
?. [Part 3 - NextGen library prep]
Mix by pipetting 40 µl 10X.
?. [Part 3 - NextGen library prep]
Split reaction into 2 x 25 µl reactions and run separately to reduce PCR bias.
?. [Part 3 - NextGen library prep]
In a thermal cycler: 1.  for  — Denaturation Cycle 2. Amplification Cycles — 8-12 cycles depending on input         a)  for         b)  for 3.  for  — Final extension cycle 4.  forever
98 °C
98 °C
65 °C
65 °C
4 °C
?. [Part 3 - NextGen library prep]
V. Bead cleanup of PCRAllow an aliquot of Ampure XP beads to warm to  for ~.
0 Room temperature
?. [Part 3 - NextGen library prep]
Vortex prior to addition to resuspend.
?. [Part 3 - NextGen library prep]
Make fresh 80% ethanol. ≥ 1 mL per reaction.
?. [Part 3 - NextGen library prep]
Add  to ligation reaction.1. Mix very well by pipetting.        a) slowly         b) expel final volume slowly due to viscosity 2. Allow to incubate at  for .
[Ampure XP beads]
0 Room temperature
?. [Part 3 - NextGen library prep]
Add  to ligation reaction.
[Ampure XP beads]
?. [Part 3 - NextGen library prep]
Mix very well by pipetting.        a) slowly         b) expel final volume slowly due to viscosity
?. [Part 3 - NextGen library prep]
Allow to incubate at  for .
0 Room temperature
?. [Part 3 - NextGen library prep]
Make fresh 80% ethanol. ≥ 1 mL per reaction.
?. [Part 3 - NextGen library prep]
Place on magnetic rack and allow beads to pellet.
?. [Part 3 - NextGen library prep]
Slowly remove supernate.
?. [Part 3 - NextGen library prep]
Ethanol wash (1/2)  Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Add .
[80% ethanol]
?. [Part 3 - NextGen library prep]
Incubate for  with beads still on magnet.
?. [Part 3 - NextGen library prep]
Ethanol wash (2/2)  Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Add .
[80% ethanol]
?. [Part 3 - NextGen library prep]
Incubate for  with beads still on magnet.
?. [Part 3 - NextGen library prep]
Remove all ethanol and allow beads to dry ~  with the lid open.
?. [Part 3 - NextGen library prep]
Elute DNA Add  to bead pellet.Wash buffer over beads and pipette to resuspend.Incubate at   for  .Place tube on magnet to separate beads.Pipette supernate and place in a a clean PCR tube.Incubate at  for .Place tube on magnet to separate beads.Pipette supernate and place in a a clean PCR tube.
[10 mM Tris-HCl]
0 Room temperature
?. [Part 3 - NextGen library prep]
Add  to bead pellet.
[10 mM Tris-HCl]
?. [Part 3 - NextGen library prep]
Wash buffer over beads and pipette to resuspend.
?. [Part 3 - NextGen library prep]
Incubate at  for .
0 Room temperature
?. [Part 3 - NextGen library prep]
Place tube on magnet to separate beads.
?. [Part 3 - NextGen library prep]
Pipette supernate and place in a a clean PCR tube.
?. [Part 3 - NextGen library prep]
VI. (Optional) Size selection of libraryA. Inefficient digestion or shearing of genomic DNA can result in large fragments (≥ 1000 bp) making it through library prep. B. In addition, size selection of double-digest RAD libraries can allow for customization of genomic coverage because all fragments originating from a specific RAD locus should be equal in length across samples but different from other RAD loci. C. Use agarose gel or Blue Pippin to size select libraries. At the very least clipping out any fragments greater than 1000 bp.
?. [Part 2 - RAD-tag isolation]
I. Before beginning the following steps, set a wet or dry bath to .
56 °C
?. [Part 2 - RAD-tag isolation]
II. Prepare Dynabead M280 streptavidin magnetic beads.Adjust bead volume.
?. [Part 2 - RAD-tag isolation]
2X Binding and Wash (B+W) Buffer.
[TrisHCl (pH 7.5) ]
[EDTA pH 8.0 ]
[NaCl]
?. [Part 2 - RAD-tag isolation]
Transfer  to a new 1.7 ml tube.
[Dynabeads]
?. [Part 2 - RAD-tag isolation]
Place tube in magnetic rack and remove supernatant.
?. [Part 2 - RAD-tag isolation]
Wash the beads (1/2) Mix Quick spin Remove supernate
[2X B+W buffer ]
?. [Part 2 - RAD-tag isolation]
Add .
[2X B+W buffer ]
?. [Part 2 - RAD-tag isolation]
Mix for .
?. [Part 2 - RAD-tag isolation]
Quick spin.
?. [Part 2 - RAD-tag isolation]
Remove supernate.
?. [Part 2 - RAD-tag isolation]
Wash the beads (2/2) Mix Quick spin Remove supernate
[2X B+W buffer ]
?. [Part 2 - RAD-tag isolation]
Add .
[2X B+W buffer ]
?. [Part 2 - RAD-tag isolation]
Mix for .
?. [Part 2 - RAD-tag isolation]
Quick spin.
?. [Part 2 - RAD-tag isolation]
Remove supernate.
?. [Part 2 - RAD-tag isolation]
Resuspend beads in X μL 2X B+W buffer (X = multiplex volume).
?. [Part 2 - RAD-tag isolation]
III. Bead BindingAdd resuspended beads to ~.
[multiplexed DNA]
?. [Part 2 - RAD-tag isolation]
While DNA fragments are binding, make a dilution BW buffer 2-fold in 10 mM Tris.1. Will need ~  per multiplex 2. Split dilution into 2x 1.5mL tubes and set one tube in a  wet or dry bath.
1 ml
56 °C
?. [Part 2 - RAD-tag isolation]
Incubate at  for . Mix every  by inverting.
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Quick spinRemove liquid from cap of tube Not enough to pellet beads
?. [Part 2 - RAD-tag isolation]
Place tube on magnetic rack - Wash (1/4)Resuspend beads in .
[1X B+W Buffer]
?. [Part 2 - RAD-tag isolation]
Place tube on magnetic rack - Wash (2/4)Resuspend beads in .
[1X B+W Buffer]
?. [Part 2 - RAD-tag isolation]
Place tube on magnetic rack - Wash (3/4)Resuspend beads in .
56 °C
[1X B+W Buffer]
?. [Part 2 - RAD-tag isolation]
Place tube on magnetic rack - Wash (4/4)
Resuspend beads in .
56 °C
[1X B+W Buffer]
?. [Part 2 - RAD-tag isolation]
IV. Liberate DNA from beadsDilute an aliquot of appropriate NEBuffer 10-fold to 1X.
?. [Part 2 - RAD-tag isolation]
Resuspend beads in  (NEBuffer 3.1 for PstI).
[1X restriction digest buffer]
?. [Part 2 - RAD-tag isolation]
Place on magnetic rack, remove supernate.
?. [Part 2 - RAD-tag isolation]
Resuspend beads in .
[1X digest buffer]
?. [Part 2 - RAD-tag isolation]
Transfer to PCR tube.
?. [Part 2 - RAD-tag isolation]
Add .
[PstI]
?. [Part 2 - RAD-tag isolation]
Incubate tube at  for .
37 °C
On Fishman Lab thermal cyclers, program available at THOM>CUTSMART.
Because the next step removes the enzyme, there is no need to heat inactivate.
?. [Part 2 - RAD-tag isolation]
Quick spin.
?. [Part 2 - RAD-tag isolation]
Place tube on magnetic rack, KEEP SUPERNATE!!
?. [Part 2 - RAD-tag isolation]
V. Bead cleanup of digestion reactionAllow an aliquot of Ampure XP beads to warm to  for ~.
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Vortex prior to addition to resuspend.
?. [Part 2 - RAD-tag isolation]
Add  to ligation reaction.1. Mix very well by pipetting     a) slowly     b) expel final volume slowly due to viscosity 2. Allow to incubate at  for .
[Ampure XP beads]
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Add  to ligation reaction.
[Ampure XP beads]
?. [Part 2 - RAD-tag isolation]
Mix very well by pipetting. slowly expel final volume slowly due to viscosity
?. [Part 2 - RAD-tag isolation]
Allow to incubate at  for .
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Make fresh 80% ethanol. ≥ 1 mL per reaction.
Use aliquoted ethanol in 50 ml conical tubes to limit evaporation/hydration of ethanol.
?. [Part 2 - RAD-tag isolation]
Place on magnetic rack and allow beads to pellet.
?. [Part 2 - RAD-tag isolation]
Slowly remove supernate.
?. [Part 2 - RAD-tag isolation]
Ethanol wash (1/2) Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 2 - RAD-tag isolation]
Add .
[80% ethanol ]
?. [Part 2 - RAD-tag isolation]
Incubate for  with beads still on magnet.
?. [Part 2 - RAD-tag isolation]
Ethanol wash (2/2) Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 2 - RAD-tag isolation]
Add .
[80% ethanol ]
?. [Part 2 - RAD-tag isolation]
Incubate for  with beads still on magnet.
?. [Part 2 - RAD-tag isolation]
Remove all ethanol and allow beads to dry ~  with the lid open.
?. [Part 2 - RAD-tag isolation]
Elute DNA 1. Add  to bead pellet. 2. Wash buffer over beads and pipette to resuspend.         a) Suspension will turn a pale, even brown. 3. Incubate at  for . 4. Place tube on magnet to separate beads.5. Pipette supernate and place in a clean PCR tube.
[10 mM Tris-HCl]
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Add  to bead pellet.
[10 mM Tris-HCl]
?. [Part 2 - RAD-tag isolation]
Wash buffer over beads and pipette to resuspend.
Suspension will turn a pale, even brown.
?. [Part 2 - RAD-tag isolation]
Incubate at  for .
0 Room temperature
?. [Part 2 - RAD-tag isolation]
Place tube on magnet to separate beads.
?. [Part 2 - RAD-tag isolation]
Pipette supernate and place in a clean PCR tube.
?. [Part 2 - RAD-tag isolation]
VI. Now is an okay time to stop if need be. Store eluted DNA at  overnight.
-20 °C
?. [Part 3 - NextGen library prep]
I. End-repair and A-tailingTo the liberated DNA, add: 1. 2.
Use full reactions NEBNext Ultra DNA Library Prep Kit for Illumina (NEB E7370S/L) with no modifications except A. Use 1:10 diluted adaptor. B. Especially if performing double-digest, use a molecular barcoded i5 adaptor (see Materials)
This step blunts single-stranded DNA ends and adds a single A overhang.
[NEBNext Ultra II End Prep Enzyme Mix ]
[NEBNext Ultra II End Prep Reaction Buffer]
?. [Part 3 - NextGen library prep]
Add  to the liberated DNA.
[NEBNext Ultra II End Prep Enzyme Mix ]
?. [Part 3 - NextGen library prep]
Add  to the liberated DNA.
[NEBNext Ultra II End Prep Reaction Buffer]
?. [Part 3 - NextGen library prep]
. Mix by pipetting 10x w/ a pipette set to 50 µl.
?. [Part 3 - NextGen library prep]
In a thermal cycler:  at  at Hold at
20 °C
65 °C
4 °C
On Fishman Lab thermal cyclers, use THOM>NEB_EP.
?. [Part 3 - NextGen library prep]
II. Adaptor ligationMake sure Ligation Master Mix is well-mixed prior to addition.
1. This part adds universal Illumina adaptors onto all end-prepped fragments. NEB NEXT adaptors form hairpins and contain a single uracil in the loop. The USER enzyme mix has endonuclease activity that cleaves the adaptor at the U, creating a Y-shaped adaptor for subsequent addition of oligos and amplification by PCR. 2. From NEB: “If input DNA ≤ 100 ng, use 1:10 diluted adaptor (diluted in 10 mM Tris, 10 mM NaCl)”. Efficient RAD preps will isolate ~1%-15% of genomic DNA. Good luck getting anything greater than 100 ng total from 48 samples!
?. [Part 3 - NextGen library prep]
Vortex quickly.
?. [Part 3 - NextGen library prep]
Quick spin.
?. [Part 3 - NextGen library prep]
To end-prepped sample, add: 1. 2. 3.
[NEBNext Ultra II Ligation Master Mix]
[NEBNext Ligation Enhancer]
[NEBNext Adaptor for Illumina (diluted 1:10 from stock)]
?. [Part 3 - NextGen library prep]
Add .
[NEBNext Ultra II Ligation Master Mix]
?. [Part 3 - NextGen library prep]
Add .
[NEBNext Ligation Enhancer]
?. [Part 3 - NextGen library prep]
Add .
[NEBNext Adaptor for Illumina (diluted 1:10 from stock)]
?. [Part 3 - NextGen library prep]
Mix sample by pipetting 10X with a pipette set to 80 µl.
?. [Part 3 - NextGen library prep]
Quick spin to collect any liquid from the side of the tube.
?. [Part 3 - NextGen library prep]
In a thermal cycler: 1. Either a) keep the thermal cycler lid open OR b) manually turn off heated lid 2.  at
20 °C
On Fishman Lab thermal cyclers, use THOM>NEB_LIG.
?. [Part 3 - NextGen library prep]
Add  to the ligation mixture.
[USER enzyme]
?. [Part 3 - NextGen library prep]
Mix sample by pipetting 10X with a pipette set to 80 µl.
?. [Part 3 - NextGen library prep]
In a thermal cycler with heated lid: 1.  at
37 °C
On Fishman Lab thermal cyclers, use THOM>NEBUSER.
?. [Part 3 - NextGen library prep]
III. Bead cleanup of ligation reactionAllow an aliquot of Ampure XP beads to warm to  for ~.
0 Room temperature
?. [Part 3 - NextGen library prep]
Vortex prior to addition to resuspend.
?. [Part 3 - NextGen library prep]
Make fresh 80% ethanol. ≥ 1 mL per reaction.
?. [Part 3 - NextGen library prep]
Add  to ligation reaction.1. Mix very well by pipetting.        a) slowly         b) expel final volume slowly due to viscosity 2. Allow to incubate at  for .
[Ampure XP beads]
0 Room temperature
?. [Part 3 - NextGen library prep]
Add  to ligation reaction.
[Ampure XP beads]
?. [Part 3 - NextGen library prep]
Mix very well by pipetting.        a) slowly         b) expel final volume slowly due to viscosity
?. [Part 3 - NextGen library prep]
Allow to incubate at  for .
0 Room temperature
?. [Part 3 - NextGen library prep]
Make fresh 80% ethanol. ≥ 1 mL per reaction.
?. [Part 3 - NextGen library prep]
Place on magnetic rack and allow beads to pellet.
?. [Part 3 - NextGen library prep]
Slowly remove supernate.
?. [Part 3 - NextGen library prep]
Ethanol wash (1/2)Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Add .
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Incubate for  with beads still on magnet.
?. [Part 3 - NextGen library prep]
Ethanol wash (2/2)Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Add .
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Incubate for  with beads still on magnet.
?. [Part 3 - NextGen library prep]
Remove all ethanol and allow beads to dry ~  with the lid open.
?. [Part 3 - NextGen library prep]
Elute DNA Add  to bead pellet.Wash buffer over beads and pipette to resuspend.Incubate at    for  .Place tube on magnet to separate beads. Pipette supernate and place in a a clean PCR tube.Incubate at   for .Place tube on magnet to separate beads. Pipette supernate and place in a a clean PCR tube.
[10 mM Tris-HCl]
0 Room temperature
?. [Part 3 - NextGen library prep]
Add  to bead pellet.
[10 mM Tris-HCl]
?. [Part 3 - NextGen library prep]
Wash buffer over beads and pipette to resuspend.
?. [Part 3 - NextGen library prep]
Incubate at   for .
0 Room temperature
?. [Part 3 - NextGen library prep]
Place tube on magnet to separate beads.
?. [Part 3 - NextGen library prep]
Pipette supernate and place in a a clean PCR tube.
?. [Part 3 - NextGen library prep]
IV. PCR enrichment of adaptor-ligated fragmentsTo the purified DNA, add:   — USE i5 WITH MOLECULAR BARCODE
[NEBNext Ultra II Q5 Master Mix]
[i7 index primer ]
[i5 universal primer]
?. [Part 3 - NextGen library prep]
Add  to the purified DNA.
[NEBNext Ultra II Q5 Master Mix]
?. [Part 3 - NextGen library prep]
Add  to the purified DNA.
[i7 index primer ]
?. [Part 3 - NextGen library prep]
Add  to the purified DNA. — USE i5 WITH MOLECULAR BARCODE
[i5 universal primer]
?. [Part 3 - NextGen library prep]
Mix by pipetting 40 µl 10X.
?. [Part 3 - NextGen library prep]
Split reaction into 2 x 25 µl reactions and run separately to reduce PCR bias.
?. [Part 3 - NextGen library prep]
In a thermal cycler: 1.  for  — Denaturation Cycle 2. Amplification Cycles — 8-12 cycles depending on input         a)  for         b)  for 3.  for  — Final extension cycle 4.  forever
98 °C
98 °C
65 °C
65 °C
4 °C
?. [Part 3 - NextGen library prep]
V. Bead cleanup of PCRAllow an aliquot of Ampure XP beads to warm to  for ~.
0 Room temperature
?. [Part 3 - NextGen library prep]
Vortex prior to addition to resuspend.
?. [Part 3 - NextGen library prep]
Make fresh 80% ethanol. ≥ 1 mL per reaction.
?. [Part 3 - NextGen library prep]
Add  to ligation reaction.1. Mix very well by pipetting.        a) slowly         b) expel final volume slowly due to viscosity 2. Allow to incubate at  for .
[Ampure XP beads]
0 Room temperature
?. [Part 3 - NextGen library prep]
Add  to ligation reaction.
[Ampure XP beads]
?. [Part 3 - NextGen library prep]
Mix very well by pipetting.        a) slowly         b) expel final volume slowly due to viscosity
?. [Part 3 - NextGen library prep]
Allow to incubate at  for .
0 Room temperature
?. [Part 3 - NextGen library prep]
Make fresh 80% ethanol. ≥ 1 mL per reaction.
?. [Part 3 - NextGen library prep]
Place on magnetic rack and allow beads to pellet.
?. [Part 3 - NextGen library prep]
Slowly remove supernate.
?. [Part 3 - NextGen library prep]
Ethanol wash (1/2)  Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Add .
[80% ethanol]
?. [Part 3 - NextGen library prep]
Incubate for  with beads still on magnet.
?. [Part 3 - NextGen library prep]
Ethanol wash (2/2)  Add .Incubate for  with beads still on magnet.
[80% ethanol ]
?. [Part 3 - NextGen library prep]
Add .
[80% ethanol]
?. [Part 3 - NextGen library prep]
Incubate for  with beads still on magnet.
?. [Part 3 - NextGen library prep]
Remove all ethanol and allow beads to dry ~  with the lid open.
?. [Part 3 - NextGen library prep]
Elute DNA Add  to bead pellet.Wash buffer over beads and pipette to resuspend.Incubate at   for  .Place tube on magnet to separate beads.Pipette supernate and place in a a clean PCR tube.Incubate at  for .Place tube on magnet to separate beads.Pipette supernate and place in a a clean PCR tube.
[10 mM Tris-HCl]
0 Room temperature
?. [Part 3 - NextGen library prep]
Add  to bead pellet.
[10 mM Tris-HCl]
?. [Part 3 - NextGen library prep]
Wash buffer over beads and pipette to resuspend.
?. [Part 3 - NextGen library prep]
Incubate at  for .
0 Room temperature
?. [Part 3 - NextGen library prep]
Place tube on magnet to separate beads.
?. [Part 3 - NextGen library prep]
Pipette supernate and place in a a clean PCR tube.
?. [Part 3 - NextGen library prep]
VI. (Optional) Size selection of libraryA. Inefficient digestion or shearing of genomic DNA can result in large fragments (≥ 1000 bp) making it through library prep. B. In addition, size selection of double-digest RAD libraries can allow for customization of genomic coverage because all fragments originating from a specific RAD locus should be equal in length across samples but different from other RAD loci. C. Use agarose gel or Blue Pippin to size select libraries. At the very least clipping out any fragments greater than 1000 bp.