Nanopore - Model SQK-PCS111 -cDNA-PCR Sequencing Kit

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

• Preparation time: ~160 mins + PCR
• Input requirement: 4 ng poly-A+ RNA, or 200 ng total RNA
• RT Required: Yes
• PCR required: Yes
• 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.