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 Chromium GEM-X Universal 3’ Gene Expression (V4) assay or the Chromium Next GEM Universal 3’ Gene Expression (V3.1) assay, 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 intended for use with the 10x Genomics Chromium GEM-X Universal 3’ Gene Expression (V4) assay or the Chromium Next GEM Universal 3’ Gene Expression (V3.1) assay.

For the 10x Genomics Chromium GEM-X Universal 5' Gene Expression (V3) assay or the Chromium Next GEM Universal 5' Gene Expression (V2) assay, please visit our Single-cell transcriptomics sequencing from 5’ cDNA prepared with 10x Genomics using SQK-LSK114 protocol.

For use with the 10x Genomics Visium Spatial Gene Expression (Visium V1 for Fresh Frozen) assay or the Visium HD 3' Spatial Gene Expression assay, please visit our Spatial transcriptomics sequencing from 3’ cDNA prepared with 10x Genomics using SQK-LSK114 and EXP-PCA001 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 Chromium GEM-X Universal 3’ Gene Expression (V4) assay or the Chromium Next GEM Universal 3’ Gene Expression (V3.1) assay. Please ensure you are using good quality sample input in this method. Additionally, 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 storage
Priming 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.

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.

Name	Sequence
[Btn]Fwd_3580_partial_read1_defined_for_3'_cDNA	5'-/5Biosg/CAGCACTTGCCTGT CGCTCTATCTTCCTACA CGACGCTCTTCCGATCT-3'
Rev_PR2_partial_TSO_defined_for_3'_cDNA	5'-CAGCTTTCTGTTGGTGCTGA TATTGCAAGCAGTGGTA TCAACGCAGAG-3'

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.

Flow cell	Minimum number of active pores covered by warranty
Flongle Flow Cell	50
MinION/GridION Flow Cell	800
PromethION Flow Cell	5000

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

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.

The sequencing device control and data acquisition are carried out by the MinKNOW software. Please ensure MinKNOW is installed on your computer or device. Further instructions for setting up a sequencing run can be found in the MinKNOW protocol.

We recommend setting up your sequencing run using the recommendations outlined below. All other parameters can be left to their default settings.

Below are the recommendations for MinKNOW settings:

Positions

Flow cell position: [user defined]

Experiment name: [user defined]

Flow cell type: FLO-PRO114M

Sample ID: [user defined]

Kit

Kit selection: Ligation Sequencing Kit (SQK-LSK114)

Run configuration

Sequencing and analysis

Basecalling: On
Modified bases: Off [default]
Model: High-accuracy basecaller (HAC)

Barcoding: Off [default]

Alignment: Off [default]

Adaptive sampling: Off [default]

Advanced options
Time between pore scans: 1.5 [default]
Reserve pores: On [default]

Data targets

Run limit: Stop run when sequencing reaches 72 hours

Output

Output format
.BAM: Off
.FASTQ: Off
Raw reads: On [default]
.POD5: On [default]

Filtering: On [default]
Qscore: 8 [default]
Minimum read length: 200 b

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 sequencing output files (.fastq or .bam) can be analysed using wf-single-cell, a bioinformatics workflow implemented in Nextflow that is part of Oxford Nanopore Technologies EPI2ME suite of analysis workflows. wf-single-cell is intended to be run from the command line but can also be run within the EPI2ME desktop app. wf-single-cell is available on GitHub.

To run the workflow from the command line:

nextflow run epi2me-labs/wf-single-cell \ 
   --expected_cells <CELL NUMBER> \ 
   --fastq <READS> \ 
   --kit <KIT> \ 
   --ref_genome_dir <REFERENCE> \ 
   -profile standard 

Where expected_cells is the number of target cells from your 10x Chromium run, fastq (or bam) is the location of the basecalled reads, kit is the 10x kit and version used (e.g. 3prime:v4), and ref_genome_dir is the location of the reference genome and annotation files as per 10x reference format (for more information see the 10x downloads centre).

Nextflow and either Docker or Singularity must be installed on your system to run the workflow. See EPI2ME Quick Start guide for additional information on how to run EPI2ME workflows from the command line.

Latest information and further details on how to run the workflow and additional parameters can be found in the workflow's GitHub page and documentation.

wf-single-cell produces an interactive HTML report, which includes basic QC metrics (sequencing score, read lengths), experiment summary (number of cells, mean reads per cell, etc), and diagnostic knee and saturation plots, as well as UMAP plots for visualising cell clusters. It also outputs gene and transcript expression matrices in a format compatible with popular downstream analysis tools.

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.