Information
This kit is highly recommended for users who:
- Would like to remove PCR bias
- Are exploring attributes of native RNA such as modified bases
Please note that to use this kit, you will need to purchase additional 3rd party reagents: see the "3rd Party Materials" tab for more detail.
The Direct RNA Sequencing Kit features:
The Direct RNA Sequencing Kit (SQK-RNA004) is used to prepare and sequence native RNA without conversion to cDNA. Inputs include poly(A)-tailed RNA or total RNA, such as eukaryotic mRNA and viral RNA.
This kit upgrade includes increased sequencing output and improved modal raw read accuracy on our new 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 new RNA flow cell. Other updates include fuel fix technology, allowing users to run longer experiments without the need for fuel addition during the run.
Further considerations:
In applications where Direct RNA sequencing’s key advantages (detection of modified bases and reduced bias) are not important, users may consider one of Oxford Nanopore’s cDNA kits 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 Monday to Thursday. Shipments to Canada, Norway, Korea and Japan are expedited Monday to Wednesday; with Australia and New Zealand leaving our warehouses on a Friday. Shipments to the rest of the world are made on Mondays to allow the full working week for packages to arrive.
The delivery charges are calculated when a quote is raised or during checkout. Once an order is made, the delivery ID and delivery information can be tracked in the Store.
Workflow
The Direct RNA Sequencing Kit (SQK-RNA004) 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 for stability to improve sequencing output. Sequencing adapters are then attached to the RNA-cDNA hybrid for sequencing.
What's in the box
The Direct RNA Sequencing Kit contains enough reagents for six reactions.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
|---|
| RT Adapter | RTA | Blue | 1 | 10 |
| RNA Ligation Adapter | RLA | Green | 1 | 45 |
| RNA CS | RCS | Yellow | 1 | 25 |
| Wash Buffer | WSB | Orange | 2 | 1,200 |
| RNA Elution Buffer | REB | Black | 1 | 300 |
| Library Solution | LIS | White cap, pink label | 1 | 600 |
| Sequencing Buffer | SB | Red | 1 | 700 |
| RNA Flush Tether | RFT | Pink | 1 | 200 |
| Flow Cell Flush | FCF | Clear cap, light blue label | 1 | 8,000 |
Note: The RNA CS (RCS) is the calibration 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 thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- Induro® Reverse Transcriptase (NEB, M0681)
- 10 mM dNTP solution (e.g. NEB N0447)
- NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
- T4 DNA Ligase 2M U/ml (NEB, M0202T/M)
- 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, cat # Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
Equipment:
- Hula mixer (gentle rotator mixer)
- Magnetic separator, suitable for 1.5 ml Eppendorf tubes
- 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
- Eppendorf 5424 centrifuge (or equivalent)
Note: The above are 3rd party reagents required for the standard prep. For other workflows using this kit, extra 3rd party reagents may be required.
Compatibility
The Direct RNA Sequencing Kit can be used together with:
Kits
- Flow Cell Wash Kit (EXP-WSH004)
Note: The Flow Cell Wash Kit (EXP-WSH004) can be used to remove library between flow cell washes and library re-loading. Please note, this kit is not compatible with RNA as a nuclease wash.
Flow cells
- FLO-MIN004RA
- FLO-PRO004RA
Devices
- MinION Mk1B
- MinION Mk1C
- GridION
- PromethION