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cDNA-PCR Sequencing Kit features
This kit is highly recommended for users who:
- Would like to identify and quantify full-length transcripts
- Are looking for a faster and simpler method for cDNA synthesis: ~210 minutes library prep + variable time for PCR
- Want to explore isoforms, splice variants and fusion transcripts using full-length cDNAs
- Wish to start from total RNA or have a low starting amount of RNA
- Would like to generate high amounts of cDNA data
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Introduction to cDNA-PCR protocol
This protocol outlines a targeted cDNA sequencing method using the cDNA-PCR Sequencing Kit (SQK-PCS111) and a user-defined sequence-specific primer. You can swap out the cDNA RT Adapter (CRTA) and design your own primer to target a specific RNA sequence during reverse transcription. During the strand-switching step, a UMI is incorporated before the double-stranded cDNA is amplified by PCR using primers containing rapid attachment chemistry. The rapid sequencing adapters are then added to the amplified sample.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:- Extract your RNA, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
- Ensure you have your sequencing kit, the correct equipment and third-party reagents.
- Download the software for acquiring and analysing your data.
- Check your flow cell to ensure it has enough pores for a good sequencing run.
Library preparation
You will need to:- Perform reverse transcription with sequence-specific primer.
- Using the strand-switching protocol, prepare full-length cDNAs from your RNA sample.
- Amplify the samples by PCR, adding rapid attachment primers during the PCR step.
- Ligate sequencing adapters to the PCR products.
- Prime the flow cell, and load your cDNA library into the flow cell.
Sequencing and analysis
You will need to:- Start a sequencing run using the MinKNOW software, which will collect raw data from the device and will basecall the reads.
- (Optional) Start the EPI2ME software and select a workflow for further analysis, e.g. metagenomic analysis or drug resistance mapping