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

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.

Order the following HPLC-purified oligos at 100 μM, and dilute to 10 μM in TE buffer for use in the Pre-pull-down step of the library prep.

Extra AMPure XP Beads will be required for the first steps when preparing the cDNA amplicons. From the end-prep step, the AMPure XP Beads (AXP) from the Ligation Sequencing Kit V14 (SQK-LSK114) can be used.

How to QC your input DNA

It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.

For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.

Chemical contaminants

Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.

We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.

For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.

Note: For this protocol, only PCR Primers (PRM) are required.

Note: We are in the process of reformatting our kits with single-use tubes into a bottle format.

Single-use tubes format:

Bottle format:

Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

MinKNOW can be used and set up to sequence in multiple ways:

• On a computer either direcly or remotely connected to a sequencing device.
• Directly on a PromethION 24/48 sequencing device.

For more information on using MinKNOW on a sequencing device, please see the device user manuals:

• PromethION user manual
• PromethION 2 Solo user manual

After sequencing has completed on MinKNOW, the flow cell can be reused or returned, as outlined in the Flow cell reuse and returns section.

After sequencing and basecalling, the data can be analysed, as outlined in the Downstream analysis section.

The workflow, wf-single-cell, processes the FASTQ format sequence data prepared by the MinKNOW software. The workflow screens each sequence read for 10X cell barcode information and assigns reads to a cell of origin. A subset of sequences from “true” cells are dynamically filtered on the basis of the number of assigned sequence reads. These sequences are mapped to the reference genome, and tables of both gene and transcript abundance are prepared for each cell. These "cell barcode x gene" or transcript abundance information are used to prepare the familiar UMAP plots that may show the stratification of the cell types present within the sample.

For more information on this workflow, follow the link to the GitHub documentation.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.