The cDNA-PCR Sequencing Kits enable the identification and quantification of full-length transcripts with highest output using nanopore sequencing.

• Low input amounts
• Highest sequencing yields
• Identify and quantify full-length transcripts and explore differential gene expression
• Characterize and quantify isoforms, splice variants, and fusion transcripts
• Measure poly-A tail lengths
• Multiplex ≤24 samples with the cDNA-PCR Barcoding Kit 24

The cDNA-PCR Sequencing Kit V14 and cDNA-PCR Barcoding Kit 24 V14 are used to prepare cDNA for nanopore sequencing from an input of as low as 10 ng poly(A)+ RNA. Users who do not have poly(A)+ enriched RNA can use 500 ng of total RNA but additional optimization 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 kit has been upgraded to use our Kit 14 chemistry, which includes a reduction of free sequencing adapter and improved median raw read sequencing accuracies (Q20+) with high output on our latest nanopore: R10.4.1. The flow cell priming and sequencing reagents are also included as part of the sequencing kit and have been reformulated to be compatible with the Kit 14 chemistry upgrade and R10.4.1 nanopore. Other updates include fuel fix technology, allowing users to run longer experiments without the need for fuel addition during the run.

The cDNA-PCR Sequencing Kit also includes a 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 poly(A)+ tail lengths. Additionally, a new post-PCR wash step further reduces short artifacts that are seen when using low-quality samples.

The cDNA-PCR Barcoding Kit 24 V14 contains 24 primer pairs and can be used instead of the cDNA-PCR Sequencing Kit V14 to multiplex up to 24 different RNA samples into a single flow cell, reducing the price per sample.

Sample multiplexing for cDNA-PCR Barcoding Kit 24 V14 is ≤24.