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Direct cDNA Sequencing Kit features
This kit is highly recommended for users who:
- are interested in exploring novel RNA biology
- do not wish to use PCR
- wish to preserve quantitative information in samples likely to be impacted by PCR bias
- would like full-length cDNAs
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Native Barcoding Expansion 1-12 and 13-24 features
These kits are recommended for users who:
- wish to multiplex samples to reduce price per sample
- need a PCR-free method of multiplexing to preserve additional information such as base modifications
- want to optimise their sequencing experiment for throughput
- require control over read length
- are interested in utilising upstream processes such as size selection or whole genome amplification
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Introduction to the Direct cDNA Native Barcoding protocol
This protocol describes how to sequencing barcoded cDNA using the Direct cDNA Sequencing Kit (SQK-DCS109) with the Native Barcoding Expansion 1-12 (EXP-NBD104) and/or the Native Barcoding Expansion 13-24 (EXP-NBD114).
In brief:
- Strand switching gives higher yields of cDNA strands
- Selects for full-length transcripts
- 5 hours library prep
- Ideal for splice variant and fusion transcript analysis
- Multiplex up to 24 different samples
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Extract your RNA, and check its length, quantity and purity. Alternatively, you can use already-prepared cDNA.
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 runLibrary preparation
You will need to:
- Using the strand-switching protocol, prepare full-length cDNAs from polyA+ RNA
- Ligate native barcodes to the cDNA
- Ligate sequencing adapters
- Prime the flow cell, and load your cDNA library into the flow cellSequencing and analysis
You will need to:
- Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
- Start the EPI2ME software and select a barcoding workflow