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Updated kit and protocol
We have released an updated version of this kit here.
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PCR-cDNA Barcoding Kit features
This kit is highly recommended for users who:
- would like to identify and quantify full-length transcripts
- want to explore isoforms, splice variants and fusion transcripts using full-length cDNAs
- have a low starting amount of RNA
- would like to generate high amounts of cDNA data
- wish to start from total RNA
- wish to multiplex samples to reduce price per sample
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Introduction to PCR-cDNA barcoding protocol for SQK-PCB109
This protocol describes how to carry out sequencing of barcoded cDNA samples using the PCR-cDNA Barcoding Kit (SQK-PCB109).
In brief:
- Strand switching gives higher yields of cDNA strands
- A faster and simpler method for cDNA synthesis
- 160 min library prep + variable time for strand-switching and PCR
- Ideal for splice variant and fusion transcript analysis
- Multiplex up to 12 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.
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 or total RNA
- Amplify the cDNAs produced by PCR, adding barcodes during the PCR step
- Pool the PCR products, and attach 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