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Introduction to the single-cell 3' transcriptomics protocol
This application allows the sequencing of full-length 3' cDNA transcripts, which provides a complete view of the expressed isoforms and allows detection of alternative splicing and fusion events in individual cells. Additionally, it enables the detection of SNPs anywhere in the transcript, as well as identification of cell sub-types in a population based on isoform expression levels.
This protocol describes how to carry out sequencing of cDNA from single cells using the Ligation Sequencing Kit V14 (SQK-LSK114) and the PCR Expansion (EXP-PCA001). You will need to have reverse-transcribed single-cell mRNA into cDNA using the 10X Genomics Next GEM Single Cell 3' Kit (V3.1) to then biotin tag your cDNA before PCR amplification with custom-ordered oligos. Next, pull-down of the amplicons on streptavidin beads is performed before a second PCR using the PCR Primers (PRM). Finally, a standard Ligation Sequencing Kit V14 library preparation is completed to prepare the cDNA ends for sequencing on a PromethION device.
This is an optimised protocol adapted from Lebrigand, K., Magnone, V., Barbry, P. et al. High throughput error corrected Nanopore single cell transcriptome sequencing. Nat Commun 11, 4025 (2020) to sequence full-length transcripts, deplete cDNA synthesis artifacts and to correct for strand bias.
Note: This protocol is compatible and fully supported with the 10X Genomics Next GEM Single Cell 3' Kit (V3.1) and the Visium Spatial Gene Expression Kit (V1). Other versions of the kits are not supported.
For the 10X Genomics Next GEM Single Cell 5' Kit (V2), please visit our Ligation sequencing V14 - Single-cell transcriptomics with 5' cDNA prepared using 10X Genomics on PromethION (SQK-LSK114) protocol.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 MinKNOW software for acquiring and analysing your data.
- Perform a flow cell check to ensure it has enough pores for a good sequencing run.
Library preparation
Protocol step Process Time Stop option Pre-pull down PCR Biotin tag your cDNA and amplify by PCR 60 minutes - Pull-down Pull-down the amplicons on streptavidin beads 40 minutes - Post-pull-down PCR Amplify the amplicons by PCR with PCR Primers (PRM) 50 minutes - End-prep Prepare the cDNA ends for adapter attachment 30 minutes 4°C overnight Adapter ligation and clean-up Attach sequencing adapters to the cDNA 20 minutes 4°C for short-term storage or for repeated use, such as for reloading your flow cell
–80°C for long-term storagePriming and loading the flow cell Prime the flow cell and load the prepared 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 basecall the reads.
- Analyse the data using the EPI2ME wf-single-cell pipeline.