Information
This kit is recommended for users who:
- have a limited amount of input material
- wish to multiplex samples to reduce price per sample
- 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 Barcoding Kit 24 V14 features:
| Feature | Property |
|---|
| Preparation time | 225 minutes + PCR |
| 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 | cDNA-PCR Sequencing V14 - Barcoding (SQK-PCB114.24) |
| Multiplexing options | • cDNA-PCR Barcoding Kit 24 V14 • 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 |
Barcoding or multiplexing is useful when the amount of data required per sample is less than the total amount of data that can be generated from a single flow cell; it allows a user to pool multiple samples and sequence them together, making more efficient use of the flow cell.
Multiplexing samples onto one flow cell can reduce the cost per sample for a user. Below are some worked examples:
| No barcodes | 6 barcodes | 12 barcodes | 24 barcodes |
|---|
| Flow cell price | $500 | $500 | $500 | $500 |
| Library price | $99 | $99 | $99 | $99 |
| Price per sample | $599 | $104 | $54 | $28 |
The cDNA-PCR Barcoding Kit is used to prepare cDNA for nanopore sequencing for up to 24 samples, from an input of as low as 4 ng polyadenylated RNA. Users who do not have polyadenylated enriched RNA can use 200 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 polyadenylated RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 24 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification using primers that contain 5’ tags and facilitate the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and the Rapid Sequencing Adapters are added to the pooled mix.
This kit has been upgraded to use our Kit 14 chemistry, which includes a reduction of free sequencing adapter and improved modal raw read sequencing accuracies (Q20+) with high output on our latest nanopore: R10.4.1. The flow cell priming and sequencing reagents are also included as part of the sequencing kit and have been reformulated to be compatible with the Kit 14 chemistry upgrade and R10.4.1 nanopore. Other updates include fuel fix technology, allowing users to run longer experiments without the need for fuel addition during the run.
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 seen 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 polyadenylated RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 24 primer pairs that are used to generate and then amplify double-stranded cDNA by PCR amplification using rapid attachment barcode primers. These primers contain 5’ tags and facilitate the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and Rapid Sequencing Adapters are added to the pooled mix.
What's in the box
The cDNA-PCR Barcoding Kit 24 V14 (SQK-PCB114.24) contains 24 unique barcodes and enough reagents for 6 reactions.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
|---|
| Strand Switching Primer II | SSPII | Violet | 1 | 350 |
| RT Primer | RTP | Yellow | 1 | 200 |
| cDNA RT Adapter | CRTA | Amber | 1 | 200 |
| Annealing Buffer | AB | Orange | 1 | 200 |
| Rapid Adapter | RA | Green | 1 | 15 |
| Adapter Buffer | ADB | Clear | 1 | 100 |
| Elution Buffer | EB | Black | 2 | 500 |
| Short Fragment Buffer | SFB | Clear | 4 | 7,500 |
| Sequencing Buffer | SB | Red | 1 | 700 |
| Library Beads | LIB | Pink | 1 | 600 |
| Library Solution | LIS | White cap, pink label | 1 | 600 |
| Barcode Primers 1-24 | BP01-24 | White | 24 | 10 |
| Flow Cell Tether | FCT | Purple | 1 | 200 |
| Flow Cell Flush | FCF | Clear cap, light blue label | 1 | 8,000 |