- Materials
-
- 2.6 x10^10 GC of rAAV per sample
- Native Barcoding Kit 24 V14 (SQK-NBD114.24)
- Consumables
-
- MinION and GridION Flow Cell
- PureLink™ Viral RNA/DNA Mini Kit (Thermo Fisher, 12280050)
- Qubit™ ssDNA Assay Kit (ThermoFisher, Q10212)
- Qubit 1x dsDNA HS Assay Kit (ThermoFisher, Q33230)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- DNase I (NEB, M0303)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- 50X annealing buffer (2.5 M NaCl, 500 mM Tris-HCl, pH 7.5)
- Ethanol, 100% (e.g. Fisher, 16606002)
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
-
- MinION or GridION device
- MinION and GridION Flow Cell Light Shield
- Hula mixer (gentle rotator mixer)
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Microfuge
- Magnetic rack
- Vortex mixer
- Thermal cycler
- Multichannel pipette and tips
- Qubit fluorometer (or equivalent for QC check)
- Eppendorf 5424 centrifuge (or equivalent)
- Timer
- 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
- Optional equipment
-
- Nanodrop spectrophotometer
-
This protocol requires 2.6 x10^10 GC of recombinant adeno-associate virus (rAAV) per sample.
A minimum of 2.6 x1010 GC of rAAV per sample has been trialled across six barcodes.
We recommend estimating genome copy number per ml (GC/ml) and to standardise rAAV inputs prior to DNAseI treatment. Droplet digital PCR (ddPCR) or qPCR are commonly used to quantify AAV vector genome numbers in titres.
-
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.
-
Check your flow cell
We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within three months of purchasing for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.
Flow cell Minimum number of active pores covered by warranty Flongle Flow Cell 50 MinION/GridION Flow Cell 800 PromethION Flow Cell 5000 -
Native Barcoding Kit 24 V14 (SQK-NBD114.24) contents
Note: We are in the process of reformatting our kits with single-use tubes into a bottle format and reducing the concentration of EDTA.
Single-use tubes format with higher EDTA concentration:
Higher concentration of EDTA with a clear cap.Bottle format with reduced EDTA concentration:
Reduced concentration of EDTA with a blue cap.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.
-
To maximise the use of the Native Barcoding Kits, the Native Barcode Auxiliary V14 (EXP-NBA114) and the Sequencing Auxiliary Vials V14 (EXP-AUX003) expansion packs are available.
These expansions provide extra library preparation and flow cell priming reagents to allow users to utilise any unused barcodes for those running in smaller subsets.
Both expansion packs used together will provide enough reagents for 12 reactions. For customers requiring extra EDTA to maximise the use of barcodes, we recommend using 0.25 M EDTA and adding 4 µl for library preps using the SQK-NBD114.24 kit and 2 µl for preps using the SQK-NBD114.96 kit.
Native Barcode Auxiliary V14 (EXP-NBA114) contents:
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.
Sequencing Auxiliary Vials V14 (EXP-AUX003) contents: