Information
This kit is highly recommended for:
- Exploring attributes of native RNA, such as modified bases
- Removing RT or PCR bias
- Transcripts that are difficult to reverse transcribe
Please note that to use this kit, additional 3rd party reagents will need to be purchased: see the "3rd Party Materials" tab for more detail.
Direct RNA Sequencing Kit XL features:
| Feature | Property |
|---|
| Preparation time | ~135 minutes |
| Input requirement | 300 ng poly(A) tailed RNA or 1 μg total RNA |
| PCR required | No |
| Fragmentation | No |
| Read length | RNA length |
| Associated protocols | Direct RNA sequencing XL (SQK-RNA004-XL) |
| Pack size | 48 reactions |
| Stability | Shipped at +2°C to +8°C
Long-term storage at -20°C to -30°C
The warranty for this Early Access product is 3 months from receipt by the customer |
The Direct RNA Sequencing Kit XL is used to prepare and sequence native RNA. Inputs include poly(A) tailed RNA or total RNA, such as eukaryotic mRNA and viral RNA.
This kit provides increased sequencing output and improved modal raw read accuracy on our RNA flow cell (FLO-MIN004RA and FLO-PRO004RA). The flow cell priming reagents are also included as part of the sequencing kit and have been reformulated to be compatible on the RNA flow cell. It also includes fuel fix technology, allowing longer experiments to be run without the need for fuel addition during the run.
Further considerations:
In applications where the key advantages of Direct RNA sequencing such as, detection of modified bases, and reduced PCR bias are not important, consider using one of the cDNA kits from Oxford Nanopore, which currently provide the highest output per run.
Shipping and logistics:
Flow cells and kits are shipped together in an environmentally friendly temperature-controlled shipping box.
Products are shipped to customers within the USA and EU from Monday to Thursday. Shipments to Canada, Norway, Korea, and Japan are expedited from Monday to Wednesday; with Australia, and New Zealand, shipments leave our warehouses on a Friday. Shipments to the rest of the world are dispatched on Mondays to allow a full working week for packages to arrive.
The delivery charges are calculated when a quote is raised or during checkout. Once an order is placed, the delivery ID, and delivery information can be tracked in the Store.
Workflow
The Direct RNA Sequencing Kit XL is used to prepare either poly(A) tailed RNA or total RNA for sequencing by performing reverse transcription to synthesise a secondary complementary cDNA strand which provides stability, and improves the sequencing output. Sequencing adapters are then attached to the RNA-cDNA hybrid for sequencing.
What's in the box
The Direct RNA Sequencing Kit XL contains sufficient reagents for 48 reactions.
| Name | Acronym | Cap colour | No. of vials/ bottles | Fill volume per vial/ bottle (μl) |
|---|
| RT Adapter | RTA | Blue | 1 vial | 150 |
| RNA Ligation Adapter | RLA | Green | 1 vial | 400 |
| RNA CS | RCS | Yellow | 3 vials | 25 |
| Wash Buffer | WSB | White | 1 bottle | 24,000 |
| RNA Elution Buffer | REB | White | 1 bottle | 10,000 |
| Library Solution | LIS | White cap, pink label | 2 vials | 1,800 |
| Sequencing Buffer | SB | Red | 3 vials | 1,700 |
| RNA Flush Tether | RFT | Violet | 1 vial | 1,600 |
| Flow Cell Flush | FCF | White | 4 bottles | 15,500 |
Note: The RNA CS (RCS) is the control strand and contains the Enolase II from YHR174W, extracted from the yeast Saccharomyces cerevisiae. The reference FASTA files for the yeast is available here.
3rd party materials
Consumables:
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml PCR strip tubes
- Eppendorf twin.tec® PCR Plate 96 LoBind®, semi-skirted (Eppendorf, 0030129504) with heat seals
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- Induro® Reverse Transcriptase and 5x Induro® RT Reaction Buffer (NEB, M0681)
- 10 mM dNTP solution (e.g. NEB N0447)
- NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
- T4 DNA Ligase 2M U/ml (NEB, M0202M)
- Murine RNase Inhibitor (NEB, M0314)
- Agencourt RNAClean XP beads (Beckman Coulter®, A63987)
- Qubit™ RNA HS Assay Kit (ThermoFisher Q32852)
- Qubit™ dsDNA HS Assay Kit (ThermoFisher, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
Equipment:
- Magnetic separation rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (ThermoFisher, 12027) or Magnetic separator suitable for 0.2 ml PCR tube strips, e.g. DynaMag™-PCR Magnet (ThermoFisher, 492025) or DynaMag™-96 Side Magnet (ThermoFisher, 12331D)
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Microfuge
- Vortex mixer
- Ice bucket with ice
- Timer
- Thermal cycler
- Qubit™ fluorometer (or equivalent for QC check)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
Note: The above are 3rd party reagents required for a standard prep. For other workflows using this kit, extra 3rd party reagents may be required.
Compatibility
The Direct RNA Sequencing Kit XL (SQK-RNA004-XL) can be used together with:
Kits
- Flow Cell Wash Kit (EXP-WSH004)
- Flow Cell Wash Kit XL (EXP-WSH004-XL)
Note: The DNase I present in the Flow Cell Wash Kit will not help to recover blocked pores in your flow cell following Direct RNA sequencing. Instead, it will wash off most of the library from the array and remove all adapter from the remaining sample, preventing it from being recaptured and sequenced. This will allow a subsequent library to be loaded.
Flow cells
- FLO-MIN004RA
- FLO-PRO004RA
Devices
- MinION Mk1B
- MinION Mk1C
- MinION Mk1D
- GridION
- PromethION
