Direct RNA Sequencing Kit
SQK-RNA002

Sequence RNA molecules directly and preserve base modifications

 Includes a Flow Cell Priming Kit

Information

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:

FeatureProperty
Preparation time105 minutes
Input requirement500 ng poly-A+ RNA
Reverse transcription requiredOptional
PCR requiredNo
Read lengthEqual to RNA length
Read type produced1D
Typical throughput*•••
Typical number of reads1 million full-length per flow cell on MinION/GridION; 3 million per flow cell on PromethION
Associated protocolsDirect RNA Sequencing and Sequence-specific direct RNA sequencing
Multiplexing optionsBarcoding kits in development and Flow Cell Wash Kit
Pack size6 reactions
StabilityShipped at 2-8° C and 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.

![]({{media url=) 2019 02 18 RNA002 v1 DS 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.

Protocols

Example protocol using Direct RNA Sequencing Kit - for illustration purposes only

What's in the box

The Direct RNA Sequencing Kit contains sufficient reagents to generate six sequencing libraries

dRNA tubes

The Flow Cell Priming Expansion Pack is also supplied.

FLP

3rd party materials

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)
  • NEBNext® Quick Ligation Reaction Buffer (NEB B6058)
  • T4 DNA Ligase 2M U/ml (NEB M0202)
  • 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)

Compatibility

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
  • MinION Mk 1C
  • GridION Mk1
  • PromethION alpha-beta/P24/P48