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 | ~240 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 | This kit is stable during shipping for 7 days at temperatures up to 25ºC. Upon receipt, please store the kit at -20°C to -30°C.
Oxford Nanopore Technologies deem 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:
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 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 |
