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Introduction to the cDNA-PCR Barcoding Kit 24 V14 protocol
This protocol describes how to carry out sequencing of multiple cDNA samples using a strand-switching method and the cDNA-PCR Barcoding Kit 24 V14 (SQK-PCB114.24). 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 using the Input DNA/RNA QC protocol. 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 preparation
The table below is an overview of the steps required in the library preparation, including timings and stopping points.
Library preparation step Process Time Stop option Reverse transcription and strand-switching Prepare full-length cDNA from Poly(A)+ RNA (or total RNA) with the incorporation of the UMI 170 minutes -20°C overnight Selecting for full-length transcripts by PCR Amplify the cDNA by PCR using rapid attachment barcode primers during the PCR step 40 minutes 4°C short-term storage or for repeated use, such as re-loading your flow cell.
-80°C for single-use long-term storage.Adapter ligation Attach the sequencing adapters to the to the PCR products. 5 minutes 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 cDNA library for sequencing 5 minutes 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