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
The cDNA-PCR Sequencing Kit features:
|Preparation time||~160 mins + PCR|
|Input requirement||4 ng poly-A+ RNA, or 200 ng total RNA|
|Read length||Enriched for full-length cDNA during PCR|
|Read type produced||1D|
|Associated protocols||PCR-cDNA Sequencing Kit (SQK-PCS111)|
|Multiplexing options||Flow Cell Wash Kit (EXP-WSH004)|
|Pack size||6 reactions|
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. 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.
• PCR-cDNA Sequencing Kit (SQK-PCS111)
To access the protocol, you will need to register for a Nanopore Community account.
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 µl|
|RT Primer||RTP||Yellow||1||10 µl|
|cDNA RT Adapter||CRTA||Amber||1||10 µl|
|Rapid Adapter T||RAP T||Green||1||10 µl|
|Annealing Buffer||AB||Orange||1||10 µl|
|cDNA Primer||cPRM||White cap, grey label||1||40 µl|
|Elution Buffer||EB||Black||1||500 µl|
|Short Fragment Buffer||SFB||Clear||1||1,800 µl|
|Sequencing Buffer II||SBII||Red||1||500 µl|
|Loading Beads II||LBII||Pink||1||360 µl|
|Loading Solution||LS||White cap, pink label||1||400 µl|
|Flush Buffer||FB||Blue||6||1,170 µl|
|Flush Tether||FLT||White cap, purple label||1||200 µl|
The cDNA-PCR Sequencing Kit can be used together with:
- Flow Cell Wash Kit (EXP-WSH004)
- Sequencing Auxiliary Vials (EXP-AUX110)
- MinION Mk1C
- MinION Mk1B