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This kit is highly recommended for users who:
- 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
- want to characterise and quantify isoforms, splice variants and fusion transcripts
The PCR-cDNA Sequencing Kit features:
Feature | Property |
Preparation time | 165 minutes |
Input requirement | 1 ng poly-A+ RNA (or 50 ng total RNA) |
Reverse transcription required | Yes |
PCR required | Yes |
Read length | Enriched for full-length cDNA during PCR |
Read type produced | 1D |
Expected data* | ••• |
Typical number of reads | 7–12+ million full-length mapped readsper flow cell on MinION/GridION; 50+ million mapped reads per flow cell on PromethION |
Associated protocols | cDNA-PCR Sequencing |
Multiplexing options |
PCR Barcoding Kit Flow Cell Wash Kit |
Pack size | 6 reactions |
Stability | Shipped at -20° C Long-term storage -20° C |
Description
The PCR-cDNA Sequencing Kit is used to prepare cDNA for nanopore sequencing 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. dscDNA is then generated by PCR amplification using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
Further considerations
The PCR Barcoding Kit (SQK-PBK004) can be used to multiplex up to 12 different RNA samples into a single flow cell.
Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.