Nanopore cDNA sequencing allows the exploration of gene expression dynamics, alternative splicing, and RNA biology with long-read capabilities. The process involves RNA extraction, cDNA synthesis, library preparation with unique barcodes, loading onto a nanopore sequencer, real-time sequencing of cDNA strands, base calling, and subsequent bioinformatics analysis for aligning reads, identifying gene isoforms, and quantifying gene expression.
The libraries are prepared with the PCR-cDNA Sequencing Kit that enables nanopore sequencing of cDNA from low input poly(A)+ RNA or total RNA with additional optimization. It employs a strand-switching method to select full-length transcripts and incorporates unique molecular identifiers (UMIs). The kit includes the Rapid Adapter T (RAP T) for enhanced capture and fuel fix technology for longer experiments without fuel addition. A new cDNA RT adapter and RT primer reduce overlaps during reverse transcription and allow measurement of polyA+ tail lengths.