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PCR-cDNA Sequencing 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
- would like to generate a large number of cDNA reads
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Introduction to the single-cell transcriptomics protocol
This protocol describes how to carry out sequencing of cDNA from single cells using the PCR-cDNA Sequencing Kit (SQK-PCS111). You will need to have reverse-transcribed single cell mRNA into cDNA using the 10X Genomics Next GEM Single Cell 3' Kit (V3.1).
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Have previously-prepared single-cell barcoded cDNA using the 10X Genomics Next GEM Single Cell 3' Kit (V3.1).
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:
- Biotin tag your cDNAs and amplify by PCR
- Pull down the amplicons on streptavidin beads, and amplify again by PCR
- Attach sequencing adapters to the PCR products
- 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
- Analyse the data further using a pipeline of your choice