Jun 07, 2023

Public workspaceRADSeq protocol EFGL

 Forked from BestRAD protocol
DOI
dx.doi.org/10.17504/protocols.io.kqdg39bjqg25/v1
  • 1Eagle Fish Genetics Lab
EagleFish GeneticsLab
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DOI: dx.doi.org/10.17504/protocols.io.kqdg39bjqg25/v1
Protocol Citation: EagleFish GeneticsLab 2023. RADSeq protocol EFGL. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39bjqg25/v1
Manuscript citation:

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 16, 2023
Last Modified: June 07, 2023
Protocol Integer ID: 77120
Keywords: Massively parallel sequencing, MPS, restriction-site associated DNA, RAD, sequence capture, genotyping, population genetics, Eagle Genetics Lab, EFGL, Radseq
Abstract
Modified from protocol of Sean O’Rourke and Mike Miller published in:
CITATION
Omar A. Ali, Sean M. O’Rourke, Stephen J. Amish, Mariah H. Meek, Gordon Luikart, Carson Jeffres and Michael R. Miller (2016). RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping. GENETICS.
LINK
https://doi.org/10.1534/genetics.115.183665

Attachments
Icon for BestRAD protocol v1.1.4 (working version).docx
BestRAD protocol v1....
460KB
Icon for BestRAD_library_prep_labData v1.1.xlsx
BestRAD_library_prep...
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Materials
MATERIALS
ReagentCutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S
ReagentNEBNext Multiplex Oligos for Illumina (Index Primers Set 1) - 24 rxnsNew England BiolabsCatalog #E7335S
ReagentPstI - 10,000 unitsNew England BiolabsCatalog #R0140S
ReagentBfaI - 500 unitsNew England BiolabsCatalog #R0568S
ReagentNEBNext Ultra II DNA Library Prep Kit for Illumina - 24 rxnsNew England BiolabsCatalog #E7645S
Restriction enzymes:
PstI: NEB
BfaI-HF: NEB
CutSmart buffer BestRAD plate adaptors:
BestRAD adaptors allow the addition of inline barcodes, and the isolation of RAD tags through purification by and enzymatic liberation of fragments from streptavidin beads. Well-specific Hamming barcodes (septamers in this case) are specified in an Excel spreadsheet and are not actually ordered as N’s. Top oligos are 5’-biotinylated and contain the 3’ PstI/SbfI overhang. Bottom oligos are 5’-phosphorylated to promote ligation.
Top oligo
Anatomy: biotin spacer SbfI barcode overhang
Sequence: /5Biosg/GTACGT CCTGCAGG NNNNNNN TGCA

Bottom oligo
Sequence: /5Phos/NNNNNNN CCTGCAGG ACGTAC
Ordering Specs
Integrated DNA technologies
  • Standard plate oligos
  • 25 nmol standard desalting
  • dry
  • $1859.52 total for a set of 48

Streptavidin purification: Dynabead M280 strepatividin, 2mL (GrizMart, ~ $500)
2X Binding and Wash Buffer: 1. 10 mM TrisHCl (pH 7.5) 2. 1 mM EDTA pH 8.0 3. 2 M NaCl 4. Concentrated (or dry) stocks should be available in chemical cabinet.
NextGen library prep:
NEBNext Ultra II Kit
  • includes reagents for end-repair, A-tail, ligation
  • NEB E7645S 24 rxns, GrizMart, Fisher
  • ~ $590

Oligos (12-plex)
  • Indexed oligos containing Illumina sequencing primer sequences and required for annealing to flow cell. Added via PCR to NEBNext adaptor-ligated fragments.
  • 12 barcoded i7 indexing primers
  • 1 universal (i5) oligo
  • NEBNext adaptor w/ USER enzyme
  • NEB E7335S
  • Grizmart
  • ~$110

Universal primer with molecular barcode
  • Modeled after i5 index primers from NEB #E7600 (p21 of manual)
  • N’s specify the equimolar addition of dATP, dTTP, dGTP, and dCTP during synthesis
• Not truly random. Some GC-bias in addition
• Should be sufficiently diverse to detect PCR duplicates
  • Used in place of the universal i5 oligo in NEB #E7335S
  • IDT, HPLC-cleaned - $90.50
  • Primer with molecular barcode in bold:
5'-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACN NNN NNN NAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC*T-3’
  • Compare to NEBNext i506 primer with indexing barcode in bold:
5´-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT AAT CTT AAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC*T-3´

Reaction purification and size selection:


Safety warnings
Please refer to the SDS (Safety Data Sheet) for safety warings and hazard information.
Annealing TOP/BOTTOM BestRAD adapters
Annealing TOP/BOTTOM BestRAD adapters
45m
45m
Preparing BestRAD adapters (Skip section if BestRAD adapters have been previously annealed)

Note
We have used 3 restriction enzymes with 6 base pair recognition sites (PstI, BamHI, and HindIII) and 1 enzyme with an 8 bp recognition site (SbfI) in our protocol. Adapters should be compatible with the base pair overhang that each restriction enzyme leaves. Both PstI and SbfI leave the same overhangs so the same adapters can be used with either enzyme. Adapters are ordered from Integrated DNA Technologies as single stranded top adapters in one plate and single stranded bottom adapters in another plate. They have to be re-hydrated, diluted to the proper concentration, and annealed prior to beginning the BestRAD protocol. See attached documents below for the sequences of our top and bottom adapters that we use. Note that the file format is for importing into IDTdna's Custom DNA Oligo, single-stranded DNA, 96 well plate form (https://www.idtdna.com/pages/products/custom-dna-rna/dna-oligos/custom-dna-oligos). We've ordered adapters a variety of ways, but the best/easiest is to order the adapters at 25nmole scale, standard desalting purification, PCR plate type, dry ship option, normalized yield and nmol quantity of 10. All top adapters are biotinylated.
We typically make a 1µM working concentration of annealed adapters if the restriction enzyme we are using is a 6 base-pair cutter. We make a 50nM working concentration of annealed adapters if the restriction enzyme is an 8 base-pair cutter. A 6 bp cutter will cut more frequently so we need a higher concentration of it.

Download BamHI_adapter_bottom.txtBamHI_adapter_bottom.txt Download BamHI_adapter_top.txtBamHI_adapter_top.txt
Download HindIII_adapter_top.txtHindIII_adapter_top.txt Download HindIII_adapter_bottom.txtHindIII_adapter_bottom.txt
Download Sbf_Pst_adapter_top.txtSbf_Pst_adapter_top.txt Download Sbf_Pst_adapter_bottom.txtSbf_Pst_adapter_bottom.txt

Note
If you already have a plate of diluted, annealed adapters, skip to Step 5

Equipment and supplies needed for steps 1-4:
thermalcycler (We have several different types of thermalcyclers. Here's an example of one we use often)
Equipment
Mastercycler Pro
NAME
thermalcycler
TYPE
Eppendorf
BRAND
No Longer Manufactured
SKU

Equipment
EPPENDORF SCIENTIFIC CENTRIFUGE 5804
NAME
plate centrifuge
TYPE
Eppendorf
BRAND
02-262-8153PM; discontinued
SKU

Equipment
Repeater-M4
NAME
pipette
TYPE
Eppendorf
BRAND
14-287-150
SKU

Equipment
ALPS 25 V
NAME
manual heated plate sealer
TYPE
Thermo Scientific
BRAND
AB-0384/110; discontinued
SKU

p10 multi-channel pipette and tips
benchtop centrifuge
1 mL Eppendorf combi-tips (Fisher Scientific; 13-683-703)
vortex
heat seals (Fisher Scientific; AB-0745)
unskirted 96 well PCR trays (Fisher Scientific; AB-0700)
Reagents needed:
Bottom adapter plate
Top adapter plate
ReagentDuplex BufferIDTCatalog #11-01-03-01
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36


Making a 100µM stock of bottom and top adapters

Assuming the bottom and top adapters were ordered dry and at a 25 nmole scale and normalized to 10nmol, first re-hydrate to 100µM by adding Amount100 µL of nuclease-free water to each well with repeater pipette and combitip

Heat seal the plate


Expected result
Now you should have a plate of Amount100 µL top adapters at 100µM concentration and a plate of Amount100 µL bottom adapters at 100µM concentration.


Making a 10uM stock of bottom and top adapters

Note

Mix the 100µM adapter plates by gently vortexing, then briefly spin the plates down Centrifigation
Use a p10 multi-channel pipette to transfer Amount10 µL of each top adapter to a new plate.
Using a repeater pipette and combitip, add Amount90 µL of nuclease-free water to the same wells of the new plate.

Heat seal the plate

Note
Even though you need to use the plates immediately in the next steps, you still need to heat seal them after every dilution so you can vortex the plate and spin it down

Repeat for the bottom adapters from step 1.4.

Heat seal the plate

Expected result
Now you should have a plate of Amount100 µL top adapters at 10µM concentration and a plate of Amount100 µL bottom adapters at 10µM concentration.

Choose between the following options:
  • If using 6 base pair cutter (PstI, BamHI, HindIII), need to make 1µM dilution of BestRAD adapters
  • If using 8 base pair cutter (SbfI), need to make 50nM dilution of BestRAD adapters
Step case

6 base pair cutter, 1µM adapters
From 22 to 212 steps

Making a 1.0µM working concentration of COMBINED bottom and top adapters

Note

Mix the 10µM adapter plates by gently vortexing, then briefly spin the plates downCentrifigation

Using a multi-channel pipette, add Amount2.5 µL of the 10µM top adapters to a new plate. Discard tips.
Using a multi-channel pipette, addAmount2.5 µL of the corresponding 10µM bottom adapters (the same well position as the top adapter plate) to the same well of the new plate. Discard tips.
Mix the duplex buffer by vortexing, then briefly spin downCentrifigation
Using a repeat pipette and a 1mL combi-tip, add Amount45 µL of duplex buffer to all wells.

Heat seal the plate

Expected result
Now you have a single plate with Amount50 µL of bottom and top adapters combined at 1µM concentration.

Annealing adapters

Run the plate of COMBINED adapters (Amount50 µL ) on a thermalcycler using the following program (In our lab, the program can be found using the menu options General -> RAD -> duplexAssay)

CHECK THE PROGRAM TO MAKE SURE IT'S CORRECT

  1. Temperature95 °C for Duration00:02:00
  2. Reduce temperature by Temperature1 °C for Duration00:01:00
  3. repeat until temperature is down to Temperature4 °C





Expected result
Now you have a single plate with Amount50 µL of bottom and top adapters combined at either 1µM concentration if you were following the 6 bp cutter steps or 50nM concentration if you were following the 8 bp cutter steps. These BestRad adapters include the barcodes for each sample


3m
Check to see if there's sufficient stock made of 2X B+W buffer (needed for step 15, size selection) and stored in 50mL conical tube at room temperature. If not, make it
Note
Equipment and supplies needed for this step:
50mL conical tube (Fisher Scientific; 12-565-271)

Reagents needed:
ReagentTris-HCl 1.0M pH 7.5Fisher ScientificCatalog #AAJ60636K2
ReagentEDTA 0.5M pH 8.0Fisher ScientificCatalog #AAJ62786AP
ReagentNaClFisher ScientificCatalog #BP358-1
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36


Add the following to a 50mL conical tube:
  1. Amount500 µL 1M Tris-HCl pH 7.5
  2. Amount100 µL 0.5M EDTA pH 8.0
  3. Amount5.844 g NaCl
  4. Add enough water to obtain Amount50 mL final volume

Note
Final concentration is 2X: 10mM Tris-HCl pH 7.5, 1mM EDTA pH 8.0, 2M NaCl

Choose from the following options:
Quantification of <48 samples
Quantification of >48 samples

Step case

<48 Samples
202 steps

DNA quantification <48 samples
DNA quantification <48 samples
Quantify DNA with Qubit

Note
Note that for sample sizes less than 48ish, we quantify each sample individually using a Qubit fluorometer. Otherwise, we use a plate reader.



Note
Equipment and supplies needed for this step:
Equipment
Qubit Fluorometer
NAME
Fluorometer to quantify DNA
TYPE
Invitrogen
BRAND
Q33226
SKU
Download 20230412_092455.jpg

Qubit assay tubes (Fisher Scientific; Q32856)
Eppendorf 2mL Snap-Cap Microcentrifuge Safe-Loc Tubes (Fisher Scientific; 05-402-7)
unskirted 96 well PCR trays (Fisher Scientific; AB-0700)

p10 pipette and tips
p200 pipette and tips

Reagents needed:
DNA tray(s)
ReagentQubit dsDNA HS assay kitFisher ScientificCatalog #Q32854
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36 if dilution necessary


Enter sample data info into “sampleData” tab of BestRAD_library_prep_labData v1.2.xlsx (attached in the DESCRIPTION tab of this protocol. It has example data for 20 samples.)
Qubit all samples with Qubit dsDNA HS assay kit.
Follow the manufacturer's protocol to make a working solution (1:200) of dye https://tools.thermofisher.com/content/sfs/manuals/Qubit_dsDNA_HS_Assay_UG.pdf
Make sure you set the sample volume and the output to be read in ng/µl, not ng/mL

Take 2 readings per sample and enter them into columns F and G in "qubitData" tab in BestRAD_library_prep_labData v1.2.xlsx. The spreadsheet has a column that will take the average. The target is 10ng/µl.
If DNA concentration is off the Qubit scale,
  1. perform a 1:5 dilution (Amount1 µL of DNA, Amount4 µL of water) in a new PCR plate
  2. Re-Qubit diluted samples
If DNA concentration is too low (less than 9ng/µl), reach out to lead biologist for replacement samples, or re-extract DNA then re-Qubit new samples
DNA Normalization
DNA Normalization
Normalize DNA to 10ng/µl

Note
Equipment and supplies needed for this step:

manual heated plate sealer
plate seals (Fisher Scientific; AB-0745)
unskirted 96 well PCR trays (Fisher Scientific; AB-0700)
p10 pipette and tips
p200 pipette and tips
vortex
plate centrifuge
Results from "Norm" tab of BestRAD_library_prep_labData v1.2.xlsx
Reagents needed:
DNA tray(s)
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36

The table in “Norm” tab of BestRAD_library_prep_labData v1.2.xlsx has been formulated to normalize all samples to 10ng/µl at a Amount30 µL final volume, and you’ll need Amount10 µL for each sample in next step.
Label a new PCR plate: Normalized DNA for [insert RADseq project name], date, initials
Pipette the amount of DNA indicated in column F of "Norm" tab of BestRAD_library_prep_labData v1.2.xlsx from the DNA tray to corresponding well position in new Normalization plate

Pipette the amount of nuclease-free water indicated in column G of "Norm" tab to corresponding well position in new Normalization plate
Heat seal plate, Mix by gently vortexing, then briefly spin the plate downCentrifigation

Expected result
Tray(s) with Amount30 µL of your samples normalized to 10ng/µl
Store at Temperature4 °C if you plan to start the library in the next couple of days,
otherwise Temperature-20 °C


Day 1 - Digestion
Day 1 - Digestion
Digest the normalized DNA with a restriction enzyme


Note
Equipment and supplies needed for this step:
thermal cycler
manual heated plate sealer
plate seals (Fisher Scientific; AB-0745)
p10 pipette and multi-channel pipette and tips
p200 pipette and tips
Repeater-M4 pipette
0.1mL combi-tip (Fisher Scientific; 13-683-700)
unskirted 96 well PCR trays (Fisher Scientific; AB-0700)
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific; 13-698-791)
vortex
plate centrifuge
timer

Reagents needed:
10µl of normalized DNA per sample
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
ReagentCutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S

1 of the following restriction enzymes
ReagentPstI - 50,000 unitsNew England BiolabsCatalog #R0140L
ReagentSbfI-HF - 2,500 unitsNew England BiolabsCatalog #R3642L
ReagentBamHI - 10,000 unitsNew England BiolabsCatalog #R0136S
ReagentHindIII - 10,000 unitsNew England BiolabsCatalog #R0104S

NOTE: All restriction enzymes' concentrations are 20,000U/µl



Thaw reagents to prep for master mix
  • Whichever restriction enzyme chosen for the project (PstI, SbfI, BamHI, or HindIII)
  • CutSmart Buffer 10X
  • Normalized DNA if it's been in the freezer
Add reagents from table below to a 1.5mL Lo-Bind tube to make a master mix. Add 5-10 to your sample size to account for pipette error. For example, if you have 20 samples, multiply the volumes per sample by 25 .


Note
Recipe above assumes 20units/µl of enzyme and 2.4 units per reaction

Vortex and briefly spin downCentrifigation

Label PCR plate "Digestion"

Using a p10 pipette or 0.1mL combitip, add Amount2 µL of master mix to each well

Using a multi-channel p10 pipette, add Amount10 µL of normalized DNA to corresponding wells

Heat seal PCR plate, vortex gently and quickly spin down. Centrifigation

Run the following thermalcycler program (In our lab, the program is "1 RE-digest" and can be found using the menu options General -> RAD)
  1. Temperature37 °C for Duration01:00:00
  2. Temperature85 °C for Duration00:30:00

CHECK THE PROGRAM TO MAKE SURE IT'S CORRECT
1h 30m
Set a timer for Duration01:15:00
Prep next step, about Duration00:15:00 prior to end of thermalcycler program

Expected result
Tray labeled "Digestion" with Amount12 µL of ~Amount100 ng of DNA per sample that has been digested with a restriction enzyme


1h 30m
Day 1 - Ligation
Day 1 - Ligation
Ligate adapters onto digested DNA

Note
Equipment and supplies needed for this step:
thermal cycler
plate centrifuge
manual heated plate sealer
plate seals (Fisher Scientific; AB-0745)
p10 pipette and multi-channel pipette and tips
p200 pipette and tips
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific; 13-698-791)
vortex
Reagents needed:
plate of annealed adapters
plate of digested DNA
ReagentCutSmart Buffer 10X (comes with restriction enzyme)New England Biolabs
Reagent100mM ATP SolutionThermofisherCatalog #R0441
ReagentT4 ligaseNew England BiolabsCatalog #M0202M
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36


  1. Thaw BestRAD adaptors (prepared in steps 1-4) and reagents for Ligation Master Mix (listed above)

gently vortex and spin down all reagentsCentrifigation

Make Ligation master mix in a 1.5mL Lo-Bind tube by combining reagents below. Multiply by the number of samples you have + 5-10 extra to account for pipette error.


Remove Digestion PCR plate from thermocycler (Step 9.9), quick spin Centrifigation

With a multi-channel p10 pipette, add Amount2 µL of annealed adaptors to each well in Digestion PCR plate, corresponding to the same well setup in the "library prep" tab of BestRAD_library_prep_labData v1.2.xlsx. Discard tip after each use.
Using a p10 pipette, add Amount2 µL of Ligation master mix to each well. Discard tip after each use.

Heat seal PCR plate, gently vortex, quick spinCentrifigation

DurationOvernight
Run thermalcycler program: (In our lab, the program is called "2 Ligation" and can be found using the menu options : General -> RAD)
  1. Temperature20 °C for Duration16:00:00
  2. Temperature80 °C for Duration00:30:00


Note
Do not let plate sit at 4°C hold for too long, recommended to proceed to next step as soon as program ends.


Expected result
Amount16 µL of digested DNA per sample with BestRad adapters attached





16h 30m
Day 2 - Sonication
Day 2 - Sonication
Prepping the Sonicator

Note
Equipment and supplies needed for this step:
Equipment
QSonica SONICATOR SYSTEM Q800R1
NAME
Sonicator
TYPE
QSonica
BRAND
discontinued
SKU
Two 1 Liter bottles for de-ionized water

Reagents needed:
de-ionized water (From Fish Health Lab)

Fetch ~Amount2 L of de-ionized water from the Fish Health Lab. Take containers with you


Connect all corresponding tubes to the machine as labeled and screw on the filter


Empty Amount1 L of de-ionized water into sonicator and start the machine.


When the filter is fully saturated, and water cycle is complete, add more de-ionized water until the level is just below the top white spout.



Proceed to next step, and let machine cool toTemperature4 °C

Day 2 - Pool samples
Day 2 - Pool samples

Note
Equipment and supplies needed for this step:
p200 pipette and tips
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific; 13-698-791)
OR
2.0mL Lo-Bind microcentrifuge tube (Fisher Scientific; 13-698-792)

Reagents needed:
"Digestion" plate of digested samples with BestRad adapters attached

Pooling samples
  • Using a p200 single channel pipette, pool all Amount16 µL of each well from Digestion PCR plate into a new 1.5mL Lo-Bind tube
  • If library contains more than 93 samples, pool into a 2.0mL Lo-Bind tube instead

Expected result
A library tube with pooled samples, volume = Amount16 µL X number of samples


Day 2 - Sonicate, precipitate, ligate library
Day 2 - Sonicate, precipitate, ligate library
purifying pooled library

Note
Equipment and supplies needed for this step:
Equipment
DynaMag Spin
NAME
Magnet for microcentrifuge tubes
TYPE
Invitrogen
BRAND
12320D
SKU
p200 pipette and tips
p1000 pipette and tips
1.5mL Lo-Bind microcentrifuge tubes (Fisher Scientific; 13-698-791)
Bioruptor Plus TPX microtubes (Fisher Scientific; C30010010-50)
5.0mL Axygen Polypropylene Snaplock non-sterile tube (Fisher Scientific; 14-568-100)
OR
15mL conical tube (Fisher Scientific; 14-959-53A)
vortex
benchtop centrifuge
Reagents needed:
tube with pooled library
ReagentAmpure XP beadsBeckman CoulterCatalog #A63881
Reagentlow TEFisher ScientificCatalog #AAJ75793AP
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
100% ethanol (laboratory grade, non-denatured!)

Note
Ampure XP beads have been aliquoted into 2 mL tubes to prevent contamination of the stock. In general, whenever they are used in the protocol, they should be vortexed thoroughly to ensure mixing

Note
if library contains >18 samples
  • Rule of thumb: Each tube should have 200-300μl of pooled samples to allow adequate room for 80% ethanol wash step
  • At the end, the combined total low TE volume of all tubes must be Amount210 µL



Using the table below, calculate the respective volumes required, or enter in number samples in "Pooling_calculator" tab in library prep spreadsheet to have volumes calculated

E.g. If N=48, then you need 3 tubes, the volume of pooled samples =Amount256 µL , volume of AMPure XP beads = Amount256 µL , volume of 80% EtOH = Amount1.024 mL , volume of low TE = Amount70 µL
Determine how many 1.5mL Lo-Bind tubes you need based on table above.
Aliquot the pooled library into the tubes equally
Add an equal volume of AMPure XP beads to pooled library amount in a 1:1 ratio

Gentle vortex, quick spin. Centrifigation

Incubate tube(s) at room temperature for Duration00:10:00

10m
During the 10 min incubation, prep 80% ethanol in 5mL tube(s) or 15ml conical
Note
e.g. to make Amount5 mL of 80%, mix Amount4 mL of 100% ethanol and Amount1 mL of nuclease-free water
For 100 samples: need Amount12 mL 100% ethanol, Amount3 mL water
For 74 samples: need Amount9.6 mL 100% ethanol, Amount2.4 mL water
For 56 samples: need Amount8 mL 100% ethanol, Amount2 mL water
For 37 samples: need Amount5.6 mL 100% ethanol, Amount1.4 mL water

The volumes above take into account the extra ~2mL of 80% ethanol needed in the rest of the protocol

Place tube(s) on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube(s) for Duration00:05:00

5m
While the tube(s) are still on the magnet, using a p200 pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard supernatant

While tube(s) are still on the magnet, add 2X total volume of 80% ethanol to the beads and incubate for Duration00:00:30 .

Note
e.g. If 48 samples: Amount256 µL of library in 3 tubes: 2X total volume = (Amount256 µL pooled library + Amount256 µL beads)*2 = Amount1024 µL 80% ethanol per tube



30s
Discard supernatant as in step 13.8

repeat step 13.9 one more time
Discard supernatant as in step 13.8
Leave tube(s) on magnet and allow any residual ethanol to evaporate by letting stand for Duration00:05:00 uncovered

5m
Take tube(s) off the magnet
Add low TE to each tube, refer to table at the beginning of this section for how much
Pipette up and down a few times to completely resuspend the bead pellet
Incubate tubes(s) at room temperature for Duration00:05:00 .

5m
Place tube(s) in magnetic stand for Duration00:05:00


Note
THE SUPERNATANT HAS THE DNA IN IT NOW. YOU WANT TO KEEP THE SUPERNATANT

5m
Using a p200 pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove the supernatant and put into a TPX tube.

Repeat for all library tubes on the magnet. Deposit all supernatants into same TPX tube
PipetteAmount210 µL of water into another TPX tube

LABEL BOTH TPX TUBES TO AVOID MIXUP!!

Expected result
1 TPX tube with Amount210 µL of pooled, digested DNA fragments
1 TPX tube with Amount210 µL of water to use as a balance in the sonicator


Sonication

Note
Equipment and supplies needed for this step:
Sonicator (prepped in step 7)
Reagents needed:
TPX tube with library
TPX tube with water as balance

Sonicate library for Duration00:04:30 at 20% capacity, and at Temperature4 °C

4m 30s
Remove tubes after sonication has completed

OK to sit in room temperature for next step.
Discard TPX vial containing water
Turn off sonicator and coolant pump
Expected result
Amount210 µL of pooled, sonicated library in tube.

Note
DNA fragments should be between 200-500bp long. Can check this on gel or Bioanalyzer here if troubleshooting needed

Some fragments now lack BestRad adapters after sonication. The next step gets rid of these


Day 2 - Bead Selection
Day 2 - Bead Selection
Prep buffers for bead binding in Step 17

Note
Equipment and supplies needed for this step:
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
p200 pipette and tips
p1000 pipette and tips
heat block
Reagents needed:
2X B+W buffer (directions on how to prepare in step 5)
ReagentCutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36


Thaw 10X CutSmart Buffer at room temperature
Check heat block to make sure it's on and set toTemperature56 °C
To a 1.5mL Lo-Bind tube add:
  • Amount25 µL 10X CutSmart Buffer
  • Amount225 µL water
To a 1.5mL Lo-Bind tube add:

  • Amount250 µL 2X B+W buffer
  • Amount250 µL water

To a 1.5mL Lo-Bind tube add:

  • Amount175 µL 2X B+W buffer
  • Amount175 µL water
Place tube from step 15.5 in heat block set to Temperature56 °C

Expected result
1 tube of Amount250 µL 1X CutSmart Buffer
1 tube of Amount500 µL 1X B +W Buffer
1 tube of warm Amount350 µL 1X B +W Buffer in heat block

Prep Dynabead M280 streptavidin magnetic beads

Note
Equipment and supplies needed for this step:
p200 pipette and tips
vortex
benchtop centrifuge
DynaMag Spin
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
Reagents needed:
2X B+W buffer made in Step 5
ReagentDynabeads M-280 StreptavidinFisher ScientificCatalog #11-206-D

Vortex stock bottle of Dynabeads and transfer Amount20 µL to new 1.5mL Lo-Bind tube

Note
Dynabeads are temperature sensitive, place beads back in fridge immediately after use

Place tube on the DynaMag Spin for Duration00:00:10

10s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the Amount20 µL supernatant

Remove tube from magnet and add Amount100 µL 2X B+W buffer
Gently vortex for Duration00:00:30 , then quick spinCentrifigation
30s
Place tube on the magnet for Duration00:00:30

30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the Amount100 µL supernatant
Repeat steps 16.4 through 16.7
Take tube off magnet, re-suspend beads in Amount200 µL of 2X B+W buffer

Vortex

Expected result
Amount200 µL of Dynabead M280 Streptavidin beads prepped for binding


Day 2 - binding DNA to beads
Day 2 - binding DNA to beads
22m
22m
Bind DNA to beads


Note
Equipment and supplies needed for this step:
p1000 pipette and tips
p200 pipette and tips
benchtop centrifuge
DynaMag Spin
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
Reagents needed:
sonicated library from Step 14.2
prepped beads from 16.9
2X B+W buffer made in Step 5
1X B+W buffer from Step 15.4
warm 1X B+W buffer from Step 15.6

Transfer all Amount210 µL of sonicated library to freshly prepped Amount200 µL Dynabeads tube from Step 16.9

Incubate for Duration00:20:00 at room temperature while gently vortexing every 2 minutes for 10 seconds (e.g 20:00 minute mark, vortex till 19:50. At 18 minutes, vortex till 17:50, etc)
20m
Quick spin Centrifigation then place tube on the DynaMag Spin for Duration00:00:30
30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the Amount410 µL supernatant
Take tube off magnet, re-suspend beads in Amount150 µL of 1X B+W buffer from Step 15.4
Mix by pipetting
Place tube on the DynaMag Spin for Duration00:00:30
30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the Amount150 µL supernatant

Repeat steps 17.5 - 17.7 2 more times



Take tube off magnet, re-suspend beads in Amount150 µL of warm 1X B+W buffer from Step 15.6

Mix by pipetting


Note
For following steps, keep warm 1X B+W buffer in heat block at Temperature56 °C when not in use

Place tube on the DynaMag Spin for Duration00:00:30
30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the Amount150 µL supernatant
Repeat steps 17.9 - 17.11 1 more time
Take tube off magnet, re-suspend beads in Amount200 µL of 2X B+W buffer

Mix by pipetting


Expected result
A tube with Amount200 µL of DNA fragments with BestRad adapters bound to the beads.


Liberate DNA from beads

Note
Equipment and supplies needed for this step:
thermal cycler
p10 pipette and tips
p200 pipette and tips
p1000 pipette and tips
plate centrifuge
DynaMag Spin
magnet stand for plate
unskirted PCR plate (Fisher Scientific; AB-0700)
manual heated plate sealer
plate seal (Fisher Scientific, AB-0745)
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
Reagents needed:
DNA and beads from Step 17.13
1X CutSmart buffer from Step 15.3
ReagentSbfI-HF - 2,500 unitsNew England BiolabsCatalog #R3642L


Re-suspend beads in Amount100 µL of 1X CutSmart buffer from Step 15.3

Mix by pipetting

Place tube on the DynaMag Spin for Duration00:00:30
30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the Amount300 µL supernatant
Repeat steps 18.1 - 18.3. Note that this time around the volume of the supernatant to remove will be Amount100 µL

Take tube off magnet, re-suspend beads with bound DNA in Amount40 µL 1X CutSmart

Mix by pipetting

Transfer all Amount40 µL beads mix into well C3 of PCR plate

Note
In our lab, we don't use any PCR tubes, only plates. This step can be done in a tube that would fit a thermalcycler.

Pipette Amount2 µL SbfI-HF (NEB R3642L) into the well

Use a p200 pipette set to 20μl to mix the solution

DO NOT VORTEX or SPIN DOWN PLATE
Heat seal PCR plate
Run thermalcycler program: 3 37hold (General -> RAD -> 3 37hold)
  • Temperature37 °C for Duration01:00:00

1h
After program has completed, give the plate a quick spinCentrifigation

Place plate on magnet in plate format for Duration00:00:30


Note
In our lab, we rigged a magnet to work on a plate. However, there are commercially available plate magnets. If you performed the incubation in a PCR tube, transfer the liquid to a tube that will fit the tube magnet stand.

30s
transfer all Amount42 µL supernatant to new 1.5mL Lo-Bind tube

Expected result
Amount42 µL of DNA fragments with BestRad adapters attached in tube



Size select DNA fragments

Note
Equipment and supplies needed for this step:

p200 pipette and tips
DynaMag Spin
unskirted PCR plate (Fisher Scientific; AB-0700)
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
Reagents needed:
80% ethanol from Step 13.6
DNA and beads from Step 18.13
Reagentlow TEFisher ScientificCatalog #AAJ75793AP
ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880


Add Amount42 µL of AMPure XP beads to Amount42 µL DNA tube

Mix by pipetting
Incubate Amount84 µL mixture at room temperature for Duration00:10:00

10m
Place tube on the DynaMag Spin for Duration00:05:00
5m
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the Amount84 µL supernatant
While still on magnet, add Amount168 µL of 80% ethanol (made the same day) to beads and incubate for Duration00:00:30

30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the Amount168 µL supernatant
Repeat wash steps 19.5 - 19.6
Leave tube on magnet and allow any residual ethanol to evaporate by letting stand for Duration00:05:00 uncapped

5m
Resuspend beads with Amount55 µL low TE

Mix by pipetting
Incubate at room temperature for Duration00:05:00

5m
Place on DynaMag Spin for Duration00:05:00

Note
NOW THE SUPERNATANT HAS THE DNA IN IT

5m
Transfer Amount55 µL supernatant to PCR plate (well C3 is preferred)

Note
If stopping is necessary, transfer supernatant to a new 1.5mL Lo-Bind tube and store in Temperature-20 °C


Expected result
Amount55 µL purified DNA fragments with BestRad adapters attached


Day 2 - Ligation
Day 2 - Ligation
Ligating NEBNext adapters to DNA fragment

Note
Equipment and supplies needed for this step:
thermal cycler
p10 pipette and tips
p200 pipette and tips
manual heated plate sealer
plate seals (Fisher Scientific, AB-0745)
plate centrifuge
vortex
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
timer
Reagents needed:
ReagentNEBNext Ultra II DNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7645L
Reagent1M Tris-HCl pH7.5Fisher ScientificCatalog #AAJ60636K2
purified DNA from Step 19.12


Note
The protocol calls for 10mM Tris-HCl pH 7.5, so make a dilution if needed
The NEBNext kit has 2 components: End Prep reagents and Ligation reagents

Thaw reagents from NEBNext End prep kit
  • Ultra II End Prep Enzyme Mix
  • Ultra II EP Buffer
Add Amount3 µL of Ultra II End Prep Enzyme Mix and Amount6.5 µL of Ultra II EP Buffer to the well of the tray with the Amount55 µL of DNA

Mix by pipetting (set p200 to 40µl)

Heat seal the plate and quick spinCentrifigation

Run thermalcycler program: 4 End Prep (General -> RAD -> End Prep)
  • Temperature20 °C for Duration00:30:00
  • Temperature65 °C for Duration00:30:00

Set a timer for 45 min
1h
While program is running, drain sonicator.
1. Disconnect the tube labeled “coolant supply” from the sonicator and remove the white nozzle end completely.
2. Turn on the pump, and tip the sonicator backwards to drain as much water as possible, then turn pump off.
3. Use paper towels to absorb all remaining water in sonicator
4. Leave lid open for a day or 2 to air dry. Unscrew the filter, empty out any remaining water and let it sit to air dry.
When timer goes off, thaw the following reagents for ligation:
  • Ultra Prep II Ligation master mix
  • NEBNext Adaptor
  • Ligation Enhancer
Dilute NEBNext adapter 1:10 in a new 1.5mL Lo-Bind tube
Amount1 µL of NEBNext Adaptor + Amount9 µL 10mM Tris HCl pH 7.5


Note
The manufacturer's protocol says to dilute the NEBnext adapter IF the DNA input is ≤ 100 ng. We don't check this and assume the adapter always needs diluting. Also, the protocol says to mix the 10mM Tris HCl pH 7.5 with 10mM NaCl, but we haven't been doing this (May, 2023).

Make Ligation master mix in a 1.5mL Lo-Bind tube by combining reagents below. Multiply by the number of samples you have + 5-10 extra to account for pipette error.


Note
The NEBNext adapter is a double stranded hairpin that will ligate to both sides of the DNA fragments. It contains the priming sites for the PCR primers in Step ______


Add master mix to well of plate with DNA (the volume should be Amount83 µL )
Mix by pipetting (set p200 to 40µl and mix)
Heat seal the plate, quick spinCentrifigation
Run thermal cycler program: 5 Adaptor Ligation (General -> RAD -> 5 Adapter Ligation
Temperature20 °C for Duration00:15:00
15m
After program has ended add Amount3 µL of NEBNext USER enzyme

Mix by pipetting as above


Note
The NEBNext adapter has a uracil base in it. The USER enzyme cleaves the hairpin adapter at the uracil so now the DNA fragments are linear and have the PCR priming sites on both sides needed in Step 22

Heat seal the plate, quick spin Centrifigation
Run thermalcycler program: 6 USER enzyme (General -> RAD -> 6 USER enzyme)
Temperature37 °C for Duration00:15:00

Expected result
A well in a PCR plate with Amount86 µL of DNA fragments that have BestRad adapters on one side with the barcodes and NEBNext adapters on both sides with the PCR priming sites


15m
Day 2 - Ampure Size Selection and Clean-Up
Day 2 - Ampure Size Selection and Clean-Up
20m
20m
Size selection and cleanup of adaptor-ligated DNA

Note
Equipment and supplies needed for this step:
p10 pipette and tips
p200 pipette and tips
1.5mL Lo-Bind microcentrifuge tubes (Fisher Scientific, 13-698-791)
DynaMag Spin
unskirted 96 well PCR tray (Fisher Scientific; AB-0700)
Reagents needed:
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
Reagentlow TEFisher ScientificCatalog #AAJ75793AP
80% ethanol


Transfer all Amount86 µL solution from 96-well unskirted PCR plate to a new 1.5mL Lo-Bind tube

Add Amount13.5 µL of nucelase-free water and Amount44 µL AMPure XP beads

mix by pipetting

Incubate at room temperature for Duration00:05:00

5m
Place tube on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube for Duration00:05:00

5m
Transfer all Amount143.5 µL supernatant to new 1.5mL Lo-Bind tube

Add Amount26 µL AMPure XP beads

mix by pipetting
Incubate at room temperature for Duration00:05:00
5m
Place tube on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube forDuration00:05:00

5m
Remove and discard supernatant (~Amount170 µL )

While still on magnet, add Amount200 µL of fresh 80% ethanol to beads and incubate for Duration00:00:30

30s
Repeat 21.9 - 21.10
Leave tube on magnet and allow any residual ethanol to evaporate by letting stand for Duration00:05:00 uncovered
5m
Take the tube off the magnet and re-suspend the beads in Amount17 µL low TE

Mix by pipetting
Incubate for Duration00:02:00 at room temperature

2m
Put tube on DynaMag Spin for Duration00:05:00

5m
Transfer Amount15 µL into a new unskirted PCR plate
OR
If stopping for the day, transfer into a new 1.5mL Lo-Bind tube, store at Temperature-20 °C

Expected result
Amount15 µL of purified DNA fragments with BestRAD and NEBNext adapters


PCR amplification
Note
Equipment and supplies needed for this step:
thermal cycler
p10 pipette and tips
200 pipette and tips
manual heated plate sealer
plate seals (Fisher Scientific, AB-0745)
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
vortex
plate centrifuge
Reagents needed:
ReagentNEBNext Multiplex Oligos for Illumina (Index Primers Set 1) - 24 rxnsNew England BiolabsCatalog #E7335S
Amount15 µL of DNA with ligated adapters from Step 21.16


Thaw reagents from NEBNext Multiplex Oligo kit (orange box) labeled: “RADseq PCR Amp”
Select an Index (i7) from the NEBNext Multiplex Oligo kit to use and record it in the "Final Library" tab in BestRAD_library_prep_labData v1.2.xlsx.
Note
The i7 MUST BE unique per library. If you are preparing more that 1 library, use different i7s.

Add the following reagents to the Amount15 µL of DNA:


Total volume is Amount50 µL

Heat seal the plate, gently vortex, quick spinCentrifigation

Run thermal cycler program: General -> RAD-> 7 NEBNext PCR 11x

Runs for about Duration00:28:00


Note
The BestRAD protocol says to perform 4-12 cycles of PCR. Currently we use 11.


28m
Transfer Amount50 µL PCR product to new 1.5mL Lo-Bind tube


Note
Do NOT stop here. Complete PCR purification below


PCR purification

Note
Equipment and supplies needed for this step:
p200 pipette and tips
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
DynaMag Spin
vortex
Reagents needed:
ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
Reagentlow TEFisher ScientificCatalog #AAJ75793AP
80% ethanol

Add Amount45 µL AMPure XP beads to the Amount50 µL of DNA from Step 22.6

mix by pipetting
Incubate at room temperature for Duration00:05:00
Place tube on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube forDuration00:05:00

Remove and discard supernatant (~Amount95 µL )

While still on magnet, add Amount200 µL of fresh 80% ethanol to beads and incubate for Duration00:00:30

Repeat 21.9 - 21.10
Leave tube on magnet and allow any residual ethanol to evaporate by letting stand for Duration00:05:00 uncovered
Take the tube off the magnet and re-suspend the beads in Amount33 µL low TE

Mix by pipetting
Incubate for Duration00:02:00 at room temperature

Put tube on DynaMag Spin for Duration00:05:00

Transfer Amount28 µL into a new 1.5mL Lo-Bind tube

Label the tube with species "Radseq", library number, date

Store at Temperature-20 °C if stopping for the day

Expected result
Amount28 µL of final, undiluted library


Day 3 - quantify library
Day 3 - quantify library
1h 3m
1h 3m
Qubit Rad library

Note
Equipment and supplies needed for this step:
Qubit fluorometer
Qubit assay tubes (Fisher Scientific, Q32856)
1.5mL or 2.0mL microcentrifuge tube
p10 pipette and tips
p200 pipette and tips
vortex
benchtop centrifuge
Reagents needed:
DNA library from Step 23.11
ReagentQubit dsDNA HS assay kitFisher ScientificCatalog #Q32854



Thaw library to room temperature
Make 1:200 diluted dye mix in a microcentrifuge tube
Amount199 µL Qubit buffer
Amount1 µL Qubit dye

Pipette Amount198 µL of diluted dye mix from Step 24.2 and Amount2 µL of the library into a qubit assay tube

Vortex thoroughly, quick spin Centrifigation

Incubate at room temperature for Duration00:03:00

3m
Put qubit assay tube in Qubit Fluorometer and take 2 readings per sample and enter them into BestRAD_library_prep_labData v1.1 spreadsheet in “Final Library” tab. The spreadsheet has a column that will take the average. The target is 4-20 ng/µl.

Note
Don't use any tube other than the qubit assay tubes in the Qubit.

Make sure you set the sample volume and the output to be read in ng/µl, not ng/mL



Expected result
Amount26 µL of your Radseq library at a concentration >= 4 ng/µl.



E-gel of library

Note
Equipment needed
p10 pipette and tips
p200 pipette and tips
parafilm
vortex
benchtop centrifuge
timer
thumb drive
Equipment
E-Gel Power Snap Electrophoresis System
NAME
e-gel electrophoresis
TYPE
Invitrogen
BRAND
G8300
SKU

Reagents needed

ReagentInvitrogen E-Gel EX Agarose Gels 1%Fisher ScientificCatalog #G402021
ReagentInvitrogen E-Gel 1 Kb Plus Express DNA ladderFisher ScientificCatalog #10-488-091
6X loading dye (comes with ladder)
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36

Thaw 1 Kb ladder, loading dye, and RAD library if needed

Gentle vortex, quick spin Centrifigation

Cut strip of parafilm, tape to benchtop if necessary
Onto parafilm, pipette Amount15 µL of 1 Kb ladder. AddAmount5 µL of nuclease free water to the ladder. Mix by pipetting

Onto parafilm, pipette Amount5 µL of the RAD library. Add Amount3 µL of loading dye and Amount12 µL of nuclease free water to the library

RETURN LIBRARY TO FREEZER
Unwrap e-gel, remove comb, remove camera (top) from the Power Snap unit and load e-gel into machine
Pipette all Amount20 µL of diluted ladder from parafilm into lane 1 of e-gel

Pipette all Amount20 µL of diluted RAD library from parafilm into lane 2 of e-gel
Pipette Amount20 µL of water into remaining empty wells of e-gel
Close lid, turn Power Snap on, select "E-gel 0.8-2%" program, push start, put camera back on Power Snap unit

Set timer for 15 min

Note
The program's duration is 26 min but the gel only needs to be run for 10-15min

When timer goes off, tap "view gel" -> adjust brightness to max ->Take picture -> Tap "Export"-> insert thumb drive into usb port -> Tap "Export"
Paste e-gel picture in the BestRAD_library_prep_labData v1.1 spreadsheet under “Final Library” tab

Crop picture to include only lane1 and lane2

Use this image to determine approximate size of library and enter it into “Final Library” tab

size of library should be ~300-500bp
Expected result
Amount21 µL of RAD library at >= 4 ng/µl and a size ~ 300- 500bp



qPCR library quantification

Note
Equipment needed
p10 pipette and tips
p200 pipette and tips
repeater M-4 pipette
1.5mL Lo-Bind microcentrifuge tubes (Fisher Scientific, 13-698-791)
Applied Biosystems MicroAmp Optical 7500 Plate (Fisher Scientific 43-168-13)
MicroAmp Optical Adhesive Film (Fisher Scientific 43-119-71)
vortex
benchtop centrifuge
0.1 mL Eppendorf combi-tips (Fisher Scientific; 13-683-700)
KimWipes
thumb drive
Equipment
7500 Real Time PCR system
NAME
qPCR
TYPE
Thermo Scientific
BRAND
discontinued
SKU

Reagents needed
ReagentNEBNext Library Quant Kit for Illumina - 500 rxnsNew England BiolabsCatalog #E7630L
ReagentInvitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
ReagentTE pH 8.0 (1X TE Solution)IDT TechnologiesCatalog #11-01-02-05

Thaw reagents in NEBNext library quantification kit and the RAD library

Gently vortex the library, quick spin Centrifigation
Check small bottle in fridge for 1X dilution of qPCR dilution buffer from NEBNext Library Quant kit. If low, make more:

Amount150 µL 10X Buffer
Amount1350 µL nuclease-free water


Make sure the master mix in the qPCR kit has been prepped. Look for the "Prep checkbox". If it hasn't, follow instructions below


Make serial dilutions in 1.5 Lo-Bind tubes as follows:

Note
The accuracy of the qPCR depends on the accuracy and precision of the preparation of the serial dilutions below. In the following steps, each time a dilution is made, it should be thoroughly mixed but you also want to avoid bubbles forming. Annoying, yes.


Note
If the Qubit score from Step 24.5 was > 25 ng/µl, use dilutions starting from 1:100,000 or 1:200,000 in qPCR

If the Qubit score was > 30 ng/µl, make addition dilutions up to 1:2,000,000 dilution

If the Qubit score was high, you may not want to run the more concentrated dilutions (1 and 2).
1. 1:1000 dilution: Amount1 µL RAD library +Amount999 µL 1X Buffer
2. 1:10,000 dilution: Amount10 µL of 1:1000 dilution + Amount90 µL 1X Buffer
3. 1:100,000 dilution:Amount10 µL of 1:10,000 dilution + Amount90 µL 1X Buffer
4. 1:200,000 dilution:Amount50 µL of 1:100,000 dilution + Amount50 µL 1X Buffer
5. 1:500,000 dilution: Amount20 µL of 1:100,000 dilution +Amount80 µL 1X Buffer
6. 1:1,000,000 dilution: Amount10 µL of 1:100,000 dilution + Amount90 µL 1X Buffer
7. 1:2,000,000 dilution: Amount50 µL of 1:1,000,000 dilution + Amount50 µL 1X Buffer



Using a 0.1mL combitip, dispense Amount8 µL of Quant Master Mix to each well of a MicroAmp Optical 7500 Plate according to plate layout
Example of plate layout - all standards and library dilutions are in triplicate
Using a p10 pipette, add Amount2 µL of standards (use standards 2-5) from qPCR kit, library dilutions and no template control (NTC; 1X Buffer) to each well according to plate layout. Discard tip after each use.

Note
Add to side of well. To avoid bubbles, DO NOT pump!

Use MicroAmp Optical Adhesive Film to seal plate (this isn't heat sealed). Use a KimWipe to press down on the seal to avoid smudges

Quick spin Centrifigation DO NOT VORTEX

Check underside of plate for dirt/lint, check wells for bubbles

Turn on 7500 qPCR system. Push to eject tray, load plate, push to close
  1. Open 7500 v2.3 software
  2. User name: GUEST
  3. Click "Template" -> Open “E7630 slow_v2” (screen will be on “Plate setup”)
  1. In left “Setup” column: Click on “Experiment Properties”
  2. Enter in Experiment name (e.g. CarpRADseq_library1_09292022)
  3. Leave everything else as is
  1. In left “Setup” column: Click on “Plate Setup”
  2. Click on “Assign Targets and Samples” tab
  3. Highlight all wells that you’re not using à Right click and select “Clear”
Click on “Save as” and save to project folder on computer
Start Run (~Duration01:00:00 )

1h
Export data.xls onto flash drive, and enter in results on NEB website to get the undiluted concentration of your RAD library
https://nebiocalculator.neb.com/#!/qPCRlibQnt

Example of Nebiocalculator – Efficiency needs to be as close to 100% as possible (+/- 10%), and R2 = 1
Note
Fragment size is the approximate band size obtained from E-gel


Dilute RAD library to 4nM using V1 = (C2*V2/V1) using 1X TE
Note
C1 = concentration of library according to qPCR
C2 = 4nM
V2 = Amount50 µL (This may change if you have a very high or low concentrated library. 50µl is appropriate if your library was ~150-250nM)


Keep original and diluted libraries at Temperature-20 °C
Expected result



Refer to "BESTRAD on NextSeq Protocol" for how to load the library

Citations
Omar A. Ali, Sean M. O’Rourke, Stephen J. Amish, Mariah H. Meek, Gordon Luikart, Carson Jeffres and Michael R. Miller. RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping
https://doi.org/10.1534/genetics.115.183665