Information
This kit is highly recommended for users who:
- have a limited amount of input material
- want to optimise their sequencing experiment for throughput
- would like to identify and quantify full-length transcripts
- are interested in differential gene expression
- want to characterise and quantify isoforms, splice variants and fusion transcripts
The PCR-cDNA Sequencing Kit features:
| Feature | Property |
|---|
| Preparation time | 165 minutes |
| 165 minutes | 1 ng poly-A+ RNA (or 50 ng total RNA) |
| Reverse transcription required | Yes |
| PCR required | Yes |
| Read length | Enriched for full-length cDNA during PCR |
| Read type produced | 1D |
| Expected data* | ••• |
| Typical number of reads | 7–12+ million full-length mapped readsper flow cell on MinION/GridION; 50+ million mapped reads per flow cell on PromethION |
| Associated protocols | cDNA-PCR Sequencing |
| Multiplexing options | PCR Barcoding Kit and Flow Cell Wash Kit |
| Pack size | 6 reactions |
| Stability | Shipped at -20° C and Long-term storage -20° C |
Description
The PCR-cDNA Sequencing Kit is used to prepare cDNA for nanopore sequencing from an input of as low as 1 ng poly-A+ RNA. Users who do not have poly-A+ enriched RNA can use 50 ng of total RNA but additional optimisation may be required.
Taking full-length poly-A+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. dscDNA is then generated by PCR amplification using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
Further considerations
The PCR Barcoding Kit (SQK-PBK004) can be used to multiplex up to 12 different RNA samples into a single flow cell.
Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.
Workflow
Taking full-length PolyA+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. dscDNA is then generated by PCR amplification using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
What's in the box
The PCR-cDNA Sequencing Kit contains sufficient reagents to generate six sequencing libraries.
The Flow Cell Priming Expansion Pack is also supplied.
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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
- Agencourt AMPure XP beads
- 10 mM dNTP solution (e.g. NEB cat # N0447)
- LongAmp Taq 2X Master Mix (e.g. NEB cat # M0287)
- Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751))
- RNaseOUT™, 40 U/μl (Life Technologies, cat # 10777019)
- Exonuclease I (NEB, cat # M0293)
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
- Pre-chilled freezer block at -20° C for 200 µl tubes (e.g. Eppendorf cat # 022510509)
Optional Equipment
- Agilent Bioanalyzer (or equivalent)
- Qubit fluorometer (or equivalent for QC check)
- Eppendorf 5424 centrifuge (or equivalent)
Compatibility
The PCR-cDNA Sequencing Kit can be used together with:
Kits
- PCR Barcoding Kit (SQK-PBK004)
- Flow Cell Wash Kit (EXP-WSH003)
Flow cells
- FLO-MINSP6
- FLO-MIN106D
- FLO-PRO002
Devices
- MinION Mk 1B
- MinION Mk 1C
- GridION Mk1
- PromethION
