Information
This kit is recommended for users who:
- wish to multiplex samples to reduce price per sample
- 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 or differential transcript usage
- want to characterise and quantify isoforms, splice variants and fusion transcripts
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 PCR-cDNA Barcoding Kit features:
| Features | PCR-cDNA Barcoding Kit |
|---|
| Preparation time | 165 mins |
| Input requirement | 1 ng poly-A+ RNA, or 50 ng total RNA |
| RT Required | Yes |
| PCR required | Yes |
| Read length | Enriched for full-length cDNA during PCR |
| Read type produced | 1D |
| Typical number of reads (on FLO-MIN106 at an average read length of 1 kb) | 7-12+ million full-length mapped reads per flow cell on MinION/GridION; 50+ million mapped reads per flow cell on PromethION |
|
| Associated protocols | PCR-cDNA sequencing - barcoding (SQK-PCB109) |
| Pack size | 6 reactions |
| Storage | 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:
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.
The PCR-cDNA Barcoding Kit is used to prepare cDNA for nanopore sequencing for up to 12 samples, 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. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates 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.
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. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates 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.
Data analysis
Data from a PCR-cDNA sequencing experiment can be analysed using a number of tutorials available in our EPI2ME Labs bioinformatic analysis suite:
- QC of cDNA reads: A workflow using pychopper providing preliminary analysis and data filtering of Oxford Nanopore Technologies cDNA read datasets
- Isoform detection: A tutorial guide for identifying full-length transcripts in your cDNA experiments and comparing them against a known annotation
- Differential gene expression: Pipeline for differential gene expression (DGE) and differential transcript usage (DTU) analysis using nanopore long reads
Protocols
PCR-cDNA sequencing - barcoding (SQK-PCB109)
To access the protocol, you will need to register for a Nanopore Community account.
What's in the box
The PCR-cDNA Barcoding Kit contains sufficient reagents to generate six sequencing libraries.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
|---|
| VN Primer | VNP | Blue | 1 | 200 |
| Strand Switching Primers | SSP | Pink | 1 | 160 |
| Rapid Adapter | RAP | Green | 1 | 10 |
| Sequencing Buffer | SQB | Red | 2 | 300 |
| Sequencing Tether | SQT | Purple | 1 | 10 |
| Loading Beads | LB | Pink | 1 | 360 |
| Elution Buffer | EB | Black | 6 | 200 |
| RNA Calibrant Strand | RCS | Yellow | 1 | 25 |
| Barcode Primers 1-12 | BP01-BP12 | Clear | 12 | 10 |
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 PCR-cDNA Barcoding Kit can be used together with:
Kits
- Flow Cell Wash Kit (EXP-WSH004)
Flow cells
Devices
- MinION Mk1B
- MinION Mk1C
- GridION
- PromethION
