Information
This kit is highly recommended for users who:
- 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
- Wish to avoid bias and artefacts introduced through PCR
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.
This is a Legacy product
This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.
The Direct cDNA Sequencing Kit is used to prepare cDNA for nanopore sequencing without using PCR. The kit recommends an input of 100 ng poly-A+RNA.
Taking full-length poly-A+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The RNA template is then digested, and the second (complementary) strand is synthesised. Sequencing adapters supplied in the Direct cDNA Sequencing Kit are then ligated onto the dscDNA.
The Direct cDNA Sequencing Kit features:
| Feature | Property |
|---|
| Preparation time | 275 minutes |
| Input requirement | 100 ng poly(A)+ RNA |
| Reverse transcription required | Yes |
| PCR required | No |
| Read length | Enriched for ful-length cDNA during second strand synthesis |
| Kit chemistry | Kit 9 |
| Typical number of reads | 5-10 million full-length per flow cell on MinION/GridION; 15-30 million per flow cell on PromethION |
| Associated protocols | • Direct cDNA sequencing
• Direct cDNA sequencing - native barcoding (SQK-DCS109 with EXP-NBD104 and EXP-NBD114) |
| Multiplexing options | • Native Barcoding Expansion Pack 1-12 (EXP-NBD104)
• Native Barcoding Expansion Pack 13-24 (EXP-NBD114)
• Flow Cell Wash Kit |
| Pack size | 6 reactions |
| Stability | Shipped at 2–8°C
Long-term storage -20°C
Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer |
Further considerations:
The Native Barcoding Expansion packs 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) can be used to multiplex up to 24 different RNA samples into a single flow cell.
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
Taking full-length messenger RNA, the complementary strand is synthesised using a kit-supplied oligo adapter. The RNA is then degraded, and the second (complementary) strand synthesised. Adapters supplied in the Direct cDNA Sequencing Kit are then ligated onto the cDNA, which introduce the components needed for the cDNA strands to enter the pore. One strand of the duplex is sequenced at a time, producing 1D reads.
What's in the box
The Direct cDNA Sequencing Kit contains sufficient reagents to generate six sequencing libraries.
| Name | Acronym | Cap colour | No. of vial | Fill volume per vial (μl) |
|---|
| VN Primer | VNP | Blue | 1 | 200 |
| Strand Switching Primer | SSP | Pink | 1 | 160 |
| PR2 Primer | PR2 | Yellow | 1 | 160 |
| Adapter Mix | AMX | Green | 1 | 40 |
| Wash Buffer | WSB | Orange | 3 | 1,000 |
| Elution Buffer | EB | Black | 1 | 200 |
| Sequencing Buffer | SQB | Red | 2 | 300 |
| Sequencing Tether | SQT | Purple | 1 | 10 |
| Loading Beads | LB | Pink | 1 | 360 |
The Flow Cell Priming Kit is also supplied.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
|---|
| Flush Buffer | FB | Blue | 6 | 1,170 |
| Flush Tether | FLT | Purple | 1 | 200 |
Compatibility
The Direct cDNA Sequencing Kit can be used together with:
Kits
- Native Barcoding Expansion 1-12 (EXP-NBD104)
- Native Barcoding Expansion 13-24 (EXP-NBD114)
- Flow Cell Wash Kit (EXP-WSH004)
Flow cells
Devices
- MinION Mk1B
- MinION Mk1C
- GridION
- PromethION