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RNA and cDNA sequencing kits
These are the previously available RNA and cDNA kits.
- cDNA-PCR Sequencing Kit (SQK-PCS109)
- cDNA-PCR Barcoding Kit (SQK-PCS111)
- Direct cDNA Sequencing Kit (SQK-DCS109)
- PCR-cDNA Barcoding Kit (SQK-PCB109)
- PCR-cDNA Barcoding Kit (SQK-PCB111.24)
- Direct RNA Sequencing Kit (SQK-RNA002)
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Direct RNA Sequencing Kit
The Direct RNA Sequencing Kit (SQK-RNA002) is used to prepare any RNA with a 3’ poly(A) tail for sequencing. Other possible RNA input includes eukaryotic mRNA, viral RNA with a poly(A) tail, or any RNA prepared with a poly(A)-tailing kit. For RNA without a poly(A) tail, users can follow simple instructions to design their own custom adapter to ligate a specific terminal 3’ sequence like the 3’ of tRNA or 16S rRNA. We recommend an input of ~500 ng of poly(A) RNA template of >200 nucleotides in length, with no upper limit.
Note: Only the RNA strand, not the RT strand, is sequenced.
Only the native RNA passes through the nanopore, meaning the read length reflects the length of the RNA molecules in the sample. The first step of the workflow requires the ligation of a reverse transcription adapter which ligates to the 3’ poly(A) tail on the RNA template molecule. Next, the optional reverse transcription step can be carried out to generate cDNA for stabilisation against RNA secondary structure formation. However, only the RNA strand is sequenced. If reverse transcription is bypassed, the workflow is shortened to 30 minutes but there is a reduction in sequencing output which 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 and the library is ready for sequencing.
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cDNA-PCR Sequencing Kit
The cDNA-PCR Sequencing Kit (SQK-PCS111) has been to Kit 11 chemistry, which requires starting inputs of 4 ng poly(A) RNA or 200 ng of total RNA. We have included a new cDNA RT adapter (CRTA), Annealing Buffer (AB) and RT primer (RTP) 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. The Kit 11 upgrade includes a new Rapid Adapter T (RAP T), with two key features which includes higher capture rate and fuel fix technology for longer experiments without the need for fuel addition during the run.
Note: Reverse transcriptase inhibits downstream PCR. Therefore, 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.
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 unique sequence (UMI) is added during reverse transcription, allowing strand switching to occur and generates a full-length cDNA strand with universal sequences on both ends. Next, PCR amplification is performed using primers with 5' tags and rapid attachment groups on the ends for adding the Rapid Sequencing Adapter (RAP T).
This kit can be used to select specific transcripts if one or both ends of the target 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 end 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 perform 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 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-PCB111.24) is the barcoding version of this kit which can be found in the Barcoding kits section.
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Direct cDNA Sequencing Kit
This kit is used to prepare cDNA for nanopore sequencing without using PCR. Preparing a library using the Direct cDNA Sequencing Kit (SQK-DCS109) takes approximately two hours longer than the cDNA-PCR Sequencing Kit (SQK-PCS109), and requires more third-party reagents. This is a PCR-free protocol and requires a higher input of 100 ng of poly(A)-tailed RNA but prevents PCR bias or polymerase error. However, data output is lower compared to the PCR version (PCR-cDNA Sequencing Kit). We recommend starting with 100 ng poly(A)-tailed RNA, or 70-200 ng of already prepared cDNA.
The first step of the workflow starts with annealing the reverse transcription primer and adding the strand-switching primer before performing reverse transcription to allow strand-switching to occur. An RNase cocktail is used to remove the RNA template strands before performing a second strand synthesis. This generates double-stranded cDNA which is treated similarly to gDNA. The fragment ends are dA-tailed before the dT-tailed sequencing adapter is ligated onto the fragments for sequencing on a flow cell.