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Direct RNA Sequencing Kit features
This kit is highly recommended for users who:
- are exploring attributes of native RNA such as modified bases.
- would like to remove RT or PCR bias.
- have transcripts that are difficult to reverse transcribe.
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Introduction to the Direct RNA Control Experiment protocol
This protocol describes how to carry out a Direct RNA Sequencing Control Experiment using the Direct RNA Sequencing Kit (SQK-RNA004). Using the RNA Control Strand (RCS), a second complementary cDNA strand is synthesised for stability by reverse transcription. Sequencing adapters are then attached to the RNA-cDNA hybrid for sequencing on either MinION or PromethION RNA Flow Cells (FLO-MIN004RA / FLO-PRO004RA respectively). Please note, the complementary cDNA strand is not sequenced, but improves the RNA sequencing output.
The control experiment can be used to become familiar with nanopore sequencing technology before preparing your own library or for troubleshooting purposes. The control experiment uses the RNA Control Strand (RCS) supplied in the kit.
Steps in the sequencing workflow:
Prepare for your experiment:
You will need to:
- Ensure you have your sequencing kit, the correct equipment and third-party reagents
- Download the MinKNOW software for acquiring and analysing your data
- Check your flow cell to ensure it has enough pores for a good sequencing run
Library preparation
The table below is an overview of the steps required in the library preparation, including timings and stopping points.
Library preparation Process Time Stop option Reverse transcription Synthesise the complementary strand of the RNA ~85 minutes At this stage the RT-RNA can be stored at -80°C for later use.
Please note, this is the only pause point in this protocol.Adapter ligation and clean-up Attach the sequencing adapters to the RNA-cDNA hybrid ends 45 minutes Attach sequencing adapters to the ends of the RNA-cDNA hybrid.
We strongly recommend sequencing your library as soon as it is adapted.Priming and loading the flow cell Prime the flow cell and load the prepared library for sequencing 5 minutes Sequencing
You will need to:
- Start a sequencing run using the MinKNOW software, which will collect raw data from the device and basecall the reads