Strand-switching protocols for preparation of 1D and 2D cDNA libraries

The Ligation Sequencing Kit contains a protocol for 1D cDNA sequencing.
Following first-strand synthesis, reverse transcriptases tend to add 1–3 non-templated Cs to the 3’ end of the cDNA strand, and by including an additional primer in the reaction which anneals to the non- templated Cs, we can make the RT enzyme switch templates, to extend opposite this primer. This incorporates a PCR-priming sequence. Following PCR, sequencing adapters are attached.

Direct RNA offers native strand sequencing, quick prep, full-length transcripts

Nanopores are capable of analysing RNA as well as DNA, but in contrast to other sequencing technologies, nanopores are the only sequencing technology which can sequence RNA directly, rather than sequencing the products of reverse transcription and PCR reactions. There are several ways to prepare RNA strands for sequencing. Fig. 1 shows one method, where a cDNA strand is synthesised, but not sequenced. An adapter is attached to the RNA strand. This allows efficient threading of the RNA strand into the nanopore, and is pre-loaded with the motor protein which controls the speed of translocation of the strand.

cDNA strand switching workflow

Direct RNA workflow