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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 Sequencing protocol
This protocol describes how to carry out sequencing of native RNA using the Direct RNA Sequencing Kit (SQK-RNA004). Starting from either poly(A) tailed RNA or total RNA, 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 the complementary cDNA strand is not sequenced, but improves the RNA sequencing output.
It is recommend that a control experiment using the RNA Control Strand (RCS) is completed first to become familiar with the technology.
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(s) to ensure it has enough pores for a good sequencing run
Library preparation
You will need to:
- Synthesise the complementary strand of the RNA
- Attach sequencing adapters to the ends of the RNA-cDNA hybrid
- Prime the flow cell, and load your RNA library onto 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 basecall the reads