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Introduction to RNA and cDNA sequencing
Using nanopore technology, RNA can be directly sequenced without sequencing complementary DNA (cDNA) intermediates, enabling the exploration of the attributes of native RNA, such as base modifications. The recently released Direct RNA Sequencing Kit (SQK-RNA004) has improved sequencing outputs and accuracy on our newly released RNA flow cells.
We have also developed kits to sequence cDNA, which is a reverse-transcribed copy of native mRNA. Therefore, characteristics of cDNA can be exploited to select for fully reverse-transcribed cDNA molecules, using the cDNA-PCR Sequencing Kit V14 (SQK-PCS114) and the cDNA-PCR Barcoding Kit V14 (SQK-PCB114.24).
Our current RNA and cDNA kits available are:
- Direct RNA Sequencing Kit (SQK-RNA004)
- cDNA-PCR Sequencing Kit V14 (SQK-PCS114)
- cDNA-PCR Barcoding Kit V14 (SQK-PCB114.24)
Below, we outline the sample input requirements and library preparation workflows for our simplex kits. For information on the barcoding kits, please see the Barcoding kits section.
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Sample input recommendations
For our RNA and cDNA sequencing kits, poly(A)-tailed mRNA is required as input as the tails are used for adapter attachment and primer annealing. However, total RNA can be used as input to the library preparation as the primers and adapters used can select for the poly(A) tails. However, using total RNA requires larger amount of input, as mRNA typically does not make up a large proportion of total RNA.
To overcome the input issue with total RNA input, poly(A) polymerase can be used to polyadenylate the RNA sample, as described in the Polyadenylation of non-poly(A) transcripts using E. coli poly(A) polymerase document. Poly(A)-tailed mRNA can also be enriched using different methods, however, regardless of methods used, there may be a slight bias towards molecules with longer poly(A) tails during library preparation as longer tails may be better targets for selection protocols. Therefore, users should be mindful of which enrichment methods are most appropriate for their needs.
To ensure high-quality sample preparation, RNA samples should be free from contaminants. If total RNA is used, an RNA Integrity Number >7 is recommended before enriching the sample using poly(A) selection. A clean RNase-free environment is crucial with good RNase-cleaning practices to prevent RNA degradation. Further information is available in the RNA contaminants and RNA stability documents.
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Direct RNA Sequencing Kit
The Direct RNA Sequencing Kit (SQK-RNA004) is our recently updated RNA kit with improved outputs and accuracy compared to the previous version of this kit. Improvements of this kit include a faster motor protein, a new RNA-specific reader pore and an optimised library preparation kit.
An input of 300 ng of poly(A)-tailed RNA or 1 µg of total RNA is required. Other possible RNA inputs includes eukaryotic mRNA, viral RNA with a poly(A) tail, or any RNA prepared with a poly(A)-tailing kit.
Workflow
Note: Only the RNA strand, not the reverse transcribed strand, is sequenced.
The library preparation prepares the RNA for sequencing by ligating a reverse transcription adapter to the RNA ends before performing reverse transcription to synthesise a complementary strand. This is to stabilise the RNA against secondary structure formation. However, only the RNA strand is sequenced. If reverse transcription is bypassed, the workflow is shortened to 30 minutes but sequencing output is reduced; this is likely due to an RNA tertiary structure blocking the pores. The sequencing adapters supplied in the kit are attached to the ends of the RNA-cDNA hybrid for sequencing.
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cDNA-PCR Sequencing Kit V14
The cDNA-PCR Sequencing Kit (SQK-PCS114) is our recently updated cDNA sequencing kit and is recommended for users starting with a low input of 10 ng of poly(A)-tailed RNA or 500 ng of total RNA, avoiding the need to poly(A)-select the template molecules.
This kit can be used for the identification and quantification of full-length transcripts. Isoforms, splice variants, and fusion transcripts can also be sequenced for analysis.
The Kit 14 chemistry upgrade in this kit reduces the level of free sequencing adapter and improves sequencing accuracies and output. Other updates include the addition of a cDNA RT adapter and RT primer to reduce transcript overlaps during the reverse transcription step to enable the measurement of the poly(A) tail length and a unique molecular identifier (UMI) for the identification of splice variants.
Workflow:
The protocol starts with a reverse transcription step to prepare full-length cDNA from the input RNA and incorporates the UMI. During reverse transcription, the poly(dT) reverse transcription adapter is ligated to the 3’ terminal poly(A) tail of the template molecule. The bottom strand of the adapter is removed and a reverse transcription primer is annealed, anchoring the start of transcription to include the entire 3’ terminal poly(A) tail. Then a strand-switching primer, containing a UMI, is added during reverse transcription, allowing strand switching to occur and generate a full-length cDNA strand. This is tagged with universal sequences on both ends.
Note: The reverse transcriptase inhibits downstream PCR and the enzymes must be heat-inactivated and the reverse transcribed sample to be split across four PCR reactions to dilute the inhibitors. This is to allow the amplification of cDNA with maximum efficiency, without losing sensitivity in the next step.
Once the full-length cDNA is prepared, PCR amplification is performed and rapid attachment primers are added to the cDNA simultaneously. Finally, rapid sequencing adapters are attached to the primers for sequencing.
Using this kit, specific transcripts can be selected if one or both ends of the target cDNA are known:
- Both target ends are known: Reverse-transcribe the entire template molecule and use selective primers to anneal to both ends of the cDNA before carrying out PCR with the sequence-specific primers.
- Only one end of the target is known: Reverse-transcribe the template molecule and use selective primers to anneal to the known end of the cDNA with universal primers on the unknown end. Then carry out PCR with the sequence-specific primers for the known end of the target molecule and a universal primer for the unknown end.
A specific transcript can also be selected by altering the reverse transcription primer to replace the poly(dT) sequence with a sequence-specific primer so only the transcript of interest will be reverse-transcribed. This reverse-transcribed and strand-switched product is then amplified with the universal primers in the PCR-cDNA Sequencing Kit.
PCR-cDNA Barcoding Kit (SQK-PCB114.24) is the barcoding version of this kit and can be found in the Barcoding kits section.