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
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 features:
The PCR-cDNA Sequencing Kit is used to prepare cDNA for nanopore sequencing from an input of as low as 4 ng poly(A)+ RNA. Users who do not have poly(A)+ 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 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.
This cDNA kit (Kit 11) includes the Rapid Adapter T (RAP T), with two key features: Firstly, the adapter is higher capture, enabling lower flow cell loading amounts. Secondly, the adapter contains the fuel fix technology, enabling users to run long experiments without the need for fuel addition during the run.
The PCR-cDNA Sequencing Kit also includes a new 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 polyA+ tail lengths.
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.
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 (SQK-PCS111) 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|
|cDNA RT Adapter||CRTA||Amber||1||10|
|Rapid Adapter T||RAP T||Green||1||10|
|cDNA Primer||cPRM||White cap, grey label||1||40|
|Short Fragment Buffer||SFB||Clear||1||1,800|
|Sequencing Buffer II||SBII||Red||1||500|
|Loading Beads II||LBII||Pink||1||360|
|Loading Solution||LS||White cap, pink label||1||400|
|Flush Tether||FLT||White cap, purple label||1||200|
The cDNA-PCR Sequencing Kit can be used together with:
- Flow Cell Wash Kit (EXP-WSH004)
- Sequencing Auxiliary Vials (EXP-AUX002)
- RNA Control Expansion (EXP-RCS001)
- RAP T Expansion (EXP-RAP111)
- MinION Mk1C
- MinION Mk1B