Jul 07, 2020
RAD-sequencing
This protocol is a draft, published without a DOI.
- 1W. Szafer Institute of Botany, Polish Academy of Sciences
Protocol Citation: Tomasz Suchan 2020. RAD-sequencing. protocols.io https://protocols.io/view/rad-sequencing-pfudjnw
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: April 13, 2018
Last Modified: July 07, 2020
Protocol Integer ID: 11476
Materials
STEP MATERIALS
Q5 Hot Start High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0493L
T4 DNA LigaseNew England BiolabsCatalog #M0202
ATP Solution (100 mM)Thermo Fisher ScientificCatalog #R0441
Q5 Hot Start High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0493L
T4 DNA LigaseNew England BiolabsCatalog #M0202
ATP Solution (100 mM)Thermo Fisher ScientificCatalog #R0441
MseI - 2,500 unitsNew England BiolabsCatalog #R0525L
SbfI - 2,500 unitsNew England BiolabsCatalog #R0642L
Protocol materials
Q5 Hot Start High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0493L
T4 DNA LigaseNew England BiolabsCatalog #M0202
ATP Solution (100 mM)Thermo Fisher ScientificCatalog #R0441
Q5 Hot Start High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0493L
T4 DNA LigaseNew England BiolabsCatalog #M0202
ATP Solution (100 mM)Thermo Fisher ScientificCatalog #R0441
MseI - 2,500 unitsNew England BiolabsCatalog #R0525L
SbfI - 2,500 unitsNew England BiolabsCatalog #R0642L
MseI - 2,500 unitsNew England BiolabsCatalog #R0525L
SbfI - 2,500 unitsNew England BiolabsCatalog #R0642L
Q5 Hot Start High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0493L
T4 DNA LigaseNew England BiolabsCatalog #M0202
ATP Solution (100 mM)Thermo Fisher ScientificCatalog #R0441
Before start
Adapter preparation
- Prepare 10 uM adapters and subsequently adapt concentration if needed (e.g. dilution for rare-cutters).
- Use an annealing buffer (prepared as 10X) for annealing single strand oligonucleotides.
10x annealing buffer (100 mM Tris-HCl pH 8, 500 mM NaCl, 10mM EDTA)
For 100ml:
- 10mL 1M Tris-HCl pH 8
- 10mL 5M NaCl
- 0.3722g EDTA
- 80mL Filtered Water
To prepare a 10 uM double-stranded adapter from 100 uM stocks, combine:
- 10 uL of each adapter single-strand oligo (100 uM)
- 10 uL annealing buffer (10X)
- 70 Ul ultrapure H2O
Incubate @95 C for 5 min followed by a slow temperature decrease (thermocycler or heating block) until room temperature. If possible, prepare a day in advance.
Working concentrations:
For Mse I – 10 uM adapters are used
For SbfI, PstI and EcoRI – dilute the 10 uM adapter tenfold for a working solution 1 uM using 10 uM Tris (or Tris-EDTA) buffer.
Alternatively, concentration of adapter can also be adjusted specifically to amount of DNA analysed and enzyme cutting frequency (see also Peterson et al. 2012 for more details):
Preparation
Preparation
Prepare reagents
Restriction reaction
Restriction reaction
Prepare the restriction reaction mix for all the samples using the amounts of reagents below:
1.9 µL nuclease-free water
0.9 µL CutSmart buffer (10x)
0.1 µL MseI (10,000 U/ml)
0.1 µL SbfI (HF) (20,000 U/ml)
MseI - 2,500 unitsNew England BiolabsCatalog #R0525L
SbfI - 2,500 unitsNew England BiolabsCatalog #R0642L
Combine 6 uL of DNA with 3 uL of the restriction mix.
Incubate 3 h at 37°C, heat-kill enzymes for 20 min at 65°C
37 °C restriction reaction
03:00:00 restriction reaction
65 °C enzyme inactivation
00:20:00 enzyme inactivation
Ligation reaction
Ligation reaction
Prepare the ligation reaction mix for all the samples using the amounts of reagents below:
0.26 µL CutSmart buffer (10x)
0.12 µL ATP (100 mM)
1 µL P2 adapter
0.17 µL T4 DNA ligase (400,000 U/ml)
T4 DNA LigaseNew England BiolabsCatalog #M0202
ATP Solution (100 mM)Thermo Fisher ScientificCatalog #R0441
0.06 µL nuclease-free water
Combine 9 uL of the restriction reaction product with 1 uL of the P1 adapter.
Safety information
Remember to use a unique P1 adapter for each sample!
Add 1.6 uL of the ligation mix to each sample.
Incubate 3 h at 16 °C
16 °C ligation reaction
03:00:00
Purification
Purification
Purify the reaction using standard AMPure XP protocol using 0.8x ratio of AMPure and elute in 20 uL of nuclease-free water or 10mM Tris
Library amplification
Library amplification
Prepare the PCR reaction mix for all the samples using the amounts of reagents below:
7.24 µL nuclease-free water
4 µL Q5 buffer (5x)
0.16 µL dNTPs (25 mM)
1.2 µL Forward primer (5 uM)
1.2 µL Reverse primer (5 uM)
0.2 µL Q5 Hot Start High-Fidelity polymerase
Q5 Hot Start High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0493L
Combine 6 uL of purified ligation product with 14 uL of the PCR mix.
PCR program:
30 s at 98°C
20cycles: 20s at 98°C, 30s at 60°C, 40s at 72°C
10 min at 72°C
hold at 4°C
Verify the reactions using agarose gel electrophoresis.
Pool the samples, including the 'blank' samples.
Purify the pool using standard AMPure XP protocol using 0.8x ratio of AMPure and elute in 20 uL of nuclease-free water or 10mM Tris.
Size selection
Size selection
Perform size selection using PippinPrep instrument or by cutting the desired fragment from the gel.
Final quantification
Final quantification
Check size selection and library fragment size using gel electrophoresis or TapeStation/Bioanalyzer/Fragment Analyzer.
Quantify the amount of DNA using Qubit.
Sequencing
Sequencing
Proceed to HiSeq/MiSeq sequencing. DNA quantity and mean fragment length can be used to calculate the molarity of the library.