SQK-RNA002
Direct RNA Sequencing Kit
$599.00
Sequence RNA molecules directly and preserve base modifications
Includes a Flow Cell Priming Kit
Direct RNA
Sequence RNA molecules directly and preserve base modifications
Up to 1 million reads
PCR cDNA
Optimised for throughput
Up to 12+ million reads
Direct cDNA
No PCR bias
Up to 10 million reads
| Direct RNA Sequencing Kit | PCR cDNA Sequencing Kit | Direct cDNA Sequencing Kit | |
|---|---|---|---|
| Preparation time | 105 mins | 165 mins | 270 mins |
| Input requirement | 500 ng poly-A+ RNA | 1 ng poly-A+ or 50 ng total RNA | 100 ng poly-A+ RNA |
| RT Required | Optional | Yes | Yes |
| PCR Required | No | Yes | No |
| Read length | Equal to RNA length | Enriched for full-length cDNA | Enriched for full-length cDNA |
| Typical throughput |
Less than 1 Gb in 6 hours 1-4 Gb in 48 hours |
2-3 Gb in 6 hours 8+ Gb in 48 hours |
1-2 Gb in 6 hours 4-8 Gb in 48 hours |
| Typical number of reads | 1 million | 7 - 12+ million | 5 - 10 million |
| Multiplexing options | In development | PCR Barcoding Kit | Native Barcoding Expansion 1-12 and Native Barcoding Expansion 13-24 |
cDNA strand switching workflow
Direct RNA workflow
This kit is highly recommended for users who:
- would like to remove RT or PCR bias
- have transcripts that are difficult to reverse transcribe
- are exploring attributes of native RNA such as modified bases
The Direct RNA Sequencing Kit features:
| Feature | Property |
| Preparation time | 105 minutes |
| Input requirement | 500 ng poly-A+ RNA |
| Reverse transcription required | Optional |
| PCR required | No |
| Read length | Equal to RNA length |
| Read type produced | 1D |
| Typical throughput* | ••• |
| Typical number of reads | 1 million full-length per flow cell on MinION/GridION; 3 million per flow cell on PromethION |
| Associated protocols |
Direct RNA Sequencing Sequence-specific direct RNA sequencing |
| Multiplexing options |
Barcoding kits in development Flow Cell Wash Kit |
| Pack size | 6 reactions |
| Stability |
Shipped at 2-8° C Long-term storage -20° C |
Description
The Direct RNA Sequencing kit (SQK-RNA002) is used to prepare any RNA with a 3’ polyA tail for 1D sequencing on the Oxford Nanopore sequencing devices. Possible input RNA includes eukaryotic mRNA, viral RNA with a polyA tail, or any RNA prepared with a polyA-tailing kit. For RNA without a polyA tail, users can follow simple instructions to design their own custom adapter to ligate a specific terminal 3’ sequence like the 3’ of tRNA or 16S rRNA. The kit requires 500 ng of input RNA.
In contrast to cDNA sequencing protocols, which are used to prepare cDNA copies of RNA for sequencing, the Direct RNA kit is used to prepare RNA itself for sequencing. In Direct RNA Sequencing, only the native RNA passes through the nanopore sensor thus the read length reflects the length of the RNA molecules in the sample.
The Direct RNA Sequencing protocol contains an optional (but recommended) reverse transcription step and though the cDNA strand generated in this step is not sequenced, it significantly improves sequencing throughput.
Further Considerations
The Direct RNA Sequencing kit is new and both accuracy and throughput will continue to improve with coming kit versions. 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 because they currently provide the highest throughput per run.
Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.
Example protocol using Direct RNA Sequencing Kit - for illustration purposes only
The Direct RNA Sequencing Kit contains sufficient reagents to generate six sequencing libraries
The Flow Cell Priming Expansion Pack is also supplied.
Consumables
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, cat # 18080044)
- 10 mM dNTP solution (e.g. NEB cat # N0447)
- Concentrated T4 DNA Ligase 2M U/ml (NEB cat # M0202)
- NEBNext® Quick Ligation Reaction Buffer (NEB cat # B6058)
- Agencourt RNAClean XP beads
- Qubit RNA HS Assay Kit (ThermoFisher cat # Q32852)
- Qubit dsDNA HS Assay Kit (ThermoFisher cat # Q32851)
Equipment
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Microfuge
- Vortex mixer
- Ice bucket with ice
- Timer
- Thermal cycler
- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
Optional Equipment
- Agilent Bioanalyzer (or equivalent)
- Qubit fluorometer (or equivalent for QC check)
- Eppendorf 5424 centrifuge (or equivalent)
The Direct RNA Sequencing Kit can be used together with:
Kits
- Flow Cell Wash Kit (EXP-WSH003)
Flow cells
- FLO-MINSP6
- FLO-MIN106D
- FLO-PRO002
Devices
- MinION Mk 1B
- GridION X5
- PromethION alpha-beta/P24/P48