Information
This kit is recommended for users who:
- Have a limited amount of input material
- Want to optimise their sequencing experiment for output
- 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
This is an Early Access product. For more information about our Early Access programmes, please see this article on product release phases.
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 cDNA-PCR Sequencing Kit V14 features:
| Feature | Property |
|---|
| Preparation time | ~340 minutes |
| Input requirement | 10 ng poly(A)+ RNA or 500 ng total RNA |
| RT Required | Yes |
| PCR Required | Yes |
| Read length | Enriched for full-length cDNA during PCR |
| Kit chemistry | Kit 14 |
| Associated protocols | Please refer to the Protocols tab. |
| Multiplexing alternatives | • cDNA-PCR Barcoding Kit V14 (SQK-PCB114.24)
• Flow Cell Wash Kit |
| Pack size | 6 reactions |
| Storage | −20°C to −30°C |
| Stability | Upon receipt, immediately store kits between −20°C and −30°C until you are ready to use them.
Kits are stable for up to 10 freeze/thaw cycles.
Oxford Nanopore Technologies deems the useful life of the product to be 3 months from receipt by the customer. |
The cDNA-PCR Sequencing Kit is used to prepare cDNA for nanopore sequencing from an input of as low as 10 ng poly(A)+ RNA. Users who do not have poly(A)+ enriched RNA can use 500 ng of total RNA but additional optimisation may be required.
The protocol uses a strand switching method to select for full length transcripts, allowing the identification of splice variants, with the incorporation of unique molecular identifiers (UMI) during this step. Taking full-length poly(A)+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. Double-stranded cDNA is then generated by PCR amplification using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
The cDNA-PCR Sequencing Kit also includes a cDNA RT adapter and RT primer to prime cDNA synthesis from the end of a transcript to reduce overlaps during the reverse transcription step and to allow users to measure poly(A)+ tail lengths. Additionally, a new post-PCR wash step further reduces short artifacts that are observed when using low-quality samples.
Shipping and logistics:
Sequencing kits are sensitive to temperature. To maintain their temperature and ensure optimal results during use, consumables are shipped with cool packs. It is not unusual for the cool packs to be warm when you receive them. For optimal results, you should immediately store your new kits at their correct temperatures.
We recommend that sequencing kits should be stored between −20°C and −30°C until you are ready to use them. Kits are stable for up to 10 freeze/thaw cycles.
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 poly(A)+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. Double-stranded cDNA 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 cDNA-PCR Sequencing Kit V14 (SQK-PCS114) kit contains enough reagents for six reactions.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
|---|
| Strand Switching Primer II | SSPII | Violet | 1 | 20 |
| RT Primer | RTP | Yellow | 1 | 10 |
| cDNA RT Adapter | CRTA | Amber | 1 | 10 |
| Annealing Buffer | AB | Orange | 1 | 10 |
| Rapid Adapter | RA | Green | 1 | 15 |
| Adapter Buffer | ADB | Clear | 1 | 100 |
| cDNA Primer | cPRM | White cap, grey label | 1 | 40 |
| Elution Buffer | EB | Black | 1 | 500 |
| Short Fragment Buffer | SFB | Clear | 1 | 1,800 |
| Sequencing Buffer | SB | Red | 1 | 700 |
| Library Beads | LIB | Pink | 1 | 600 |
| Library Solution | LIS | White cap, pink label | 1 | 600 |
| Flow Cell Tether | FCT | Purple | 1 | 200 |
| Flow Cell Flush | FCF | Clear cap, light blue label | 1 | 8,000 |
