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PCR-cDNA Barcoding Kit features:
This kit is highly recommended for users who:
- wish to multiplex up to 24 samples to reduce price per sample
- 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
- have a low starting amount of RNA
- would like to generate high amounts of cDNA data
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Introduction to the PCR-cDNA Barcoding Kit protocol
This protocol describes how to carry out sequencing of multiple cDNA samples using a strand-switching method and the PCR-cDNA Barcoding Kit (SQK-PCB111.24). There are 24 unique barcodes available, allowing the user to pool up to 24 different samples in one sequencing experiment. During the strand-switching step, a UMI is incorporated, before the double-stranded cDNA is amplified by PCR using primers containing 5' tags. The amplified and barcoded samples are then pooled together and the Rapid Sequencing Adapters are added to the pooled mix.
A control experiment can be completed first using RNA Control Sample (RCS) from the RNA Control Expansion (EXP-RCS001) as your input to troubleshoot your library preparation or to become familiar with the protocol.
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 preparationYou will need to:
- Using the strand-switching protocol, prepare full-length cDNAs from Poly(A)+ RNA (or total RNA) with the incorporation of the UMI
- Amplify the cDNAs by PCR, adding rapid attachment barcode primers during the PCR step
- Attach 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 convert it into basecalled reads
- Optional: Start the EPI2ME software and select a workflow for further analysis