- Materials
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- 10 µg of high molecular weight genomic DNA or 5 x 10⁶ cells (e.g. cell culture or tissue)
- Short Fragment Eliminator (EXP-SFE001) kit
- Ligation Sequencing Kit V14 (SQK-LSK114)
- Flow Cell Wash Kit (EXP-WSH004)
- Sequencing Auxiliary Vials V14 (EXP-AUX003)
- Consumables
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- NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (cat # E7180S or E7180L).
Alternatively, you can use the NEBNext® products below:
- NEBNext FFPE Repair Mix (NEB, M6630)
- NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- Puregene Cell Kit (QIAGEN, 158043)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- 15 ml Falcon tubes
- 2 ml Eppendorf DNA LoBind tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Freshly prepared 70% ethanol in nuclease-free water
- Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
- Isopropanol, 100% (Fisher, 10723124)
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- Inoculation loop or disposable tweezers
- (Optional) TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Agilent Genomic DNA 165 kb Analysis Kit (Agilent, FP-1002-0275)
- Equipment
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- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Microfuge
- Vortex mixer
- Thermal cycler
- Centrifuge and rotor suitable for 15 ml Falcon tubes
- Incubator or water bath set at 37°C and 50°C
- Megaruptor 3 (Diagenode, B06010003)
- Wide-bore pipette tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Ice bucket with ice
- Timer
- Heating block
- Ice bucket with ice
- Agilent Femto Pulse System (or equivalent for read length QC)
- Qubit fluorometer (or equivalent for QC check)
- Optional equipment
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- Shaking heat block
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For this protocol, you will need the following input:
For this end-to-end workflow, we recommend extracting high molecular weight gDNA from 5 x 10⁶ cells (e.g. cell culture or tissue sample) using the QIAGEN Puregene Cell Kit in the Sample Preparation step.
Other extraction protocols are available but have not been tested by Oxford Nanopore Technologies.
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Input DNA
How to QC your input DNA
It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.
For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.
Chemical contaminants
Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.
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NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing
For customers new to nanopore sequencing, we recommend buying the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7180S or E7180L), which contains all the NEB reagents needed for use with the Ligation Sequencing Kit.
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Third-party reagents
We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.
For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.
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Ligation Sequencing Kit V14 (SQK-LSK114) contents
Note: We are in the process of reformatting our kits with single-use tubes into a bottle format.
Single-use tubes format:
Bottle format:
Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.
Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.
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Short Fragment Eliminator (EXP-SFE001) kit contents
Name Acronym Cap colour No. of vials Fill volume per vial (µl) Short Fragment Eliminator buffer SFE Orange 3 1,200 -
Sequencing Auxiliary Vials V14 (EXP-AUX003) contents
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Elution Buffer EB Black 2 500 Sequencing Buffer SB Red 2 700 Library Solution LIS White cap, pink label 2 600 Library Beads LIB Pink 2 600 Flow Cell Flush FCF Light blue label 2 8,000 Flow Cell Tether FCT Purple 2 200 -
Flow Cell Wash Kit (EXP-WSH004) contents
Contents Volume (µl) No. of tubes No. of uses Wash Mix (WMX) 15 1 6 Wash Diluent (DIL) 1,300 2 6 Storage Buffer (S) 1,600 2 6 - Wash Mix (WMX) contains DNase I.
- Wash Diluent (DIL) contains the exonuclease buffer that maximises activity of the DNase I.
- The Storage Buffer allows flow cells to be stored for extended periods of time.