10x Genomics single-cell transcriptomics human cell lines
This post details the release of 10x Genomics single cell cDNA sequencing of two cell lines: 293T and Jurkat. For each cell line two biological replicates were prepared, with each replicate being sequences with two PromethION Flow Cells (for a total of 8 Flow Cells). The 293T data were prepared using the Chromium GEM-X Universal 3’ Gene Expression v4 kit, while Jurkat data were prepared using the Chromium GEM-X Universal 5’ Gene Expression v3 kit.
Dataset
The dataset is available for anonymous download, without login credentials, from a public Amazon Web Services S3 bucket. The bucket is part of the Open Data on AWS project enabling sharing and analysis of a wide range of data. The data is located in the bucket at:
s3://ont-open-data/sc-gemx-2025.02
See the tutorials page for information on downloading the dataset.
You can also browse and download the files in your web browser curtesy of 42basepairs.
| Folder name | Description | Size |
|---|---|---|
| RAW | POD5 files | 15 TB |
| basecalls | BAM files | 1.1 TB |
| analysis/workflow_outputs | Workflow outputs | 589 GB |
| analysis/basic_outputs | Minimum outputs required for downstream tools | 2 GB |
Sample
| Attribute | Value |
|---|---|
| Sample Name(s) | 293t, jurkat |
| Organism | Human |
| Molecule Type | cDNA |
| Sample Type | cell culture |
| Biological replicates per sample | 2 |
| Flow cells per sample | 2 |
Preparation
Sample preparation was performed according to protocols published on the main Oxford Nanopore Technologies’ website.
| Attribute | Value |
|---|---|
| Library Prep | 10x Genomics 3’ cDNA, 10x Genomes 5’ cDNA |
| Sequencing Kit | Ligation sequencing V14 (SQK-LSK114) |
Further information such as sample storage suggestions can be found at https://nanoporetech.com/documentation/prepare.
Sequencing
Sequence data was generated using the PromethION sequencer, with the following configuration:
| Attribute | Value |
|---|---|
| Flow Cell | FLO-PRO114M |
| Device | PromethION |
| Chemistry | R10.4.1 |
| Basecall Model(s) | dna_r10.4.1_e8.2_400bps_hac@v5.0.0 |
| MinKNOW Core Version | 6.2.6 |
| PromethION release package | 24.11.8 |
using the the following 10x Genomics chemistries to prepare initial cDNA libraries:
| Attribute | Value |
|---|---|
| Preservation method | Cryopreserved |
| Chemistry | GEM-X |
| Kit | GEM-X Universal 5’ Gene Expression v3, GEM-X Universal 3’ Gene Expression v4 |
| Target number of cells | 1000 |
Basecalling was performed off device using dorado (v0.9.1).
Analyses
To perform cell demulltiplexing and UMI deduplication we used our wf-single-cell (v2.4.1) Nextflow workflow.
The reference used in the analyses and provided to the workflows --ref_genome_dir parameter is available to download from 10x Genomics.
The analysis results are located in the S3 bucket under the prefix:
s3://ont-open-data/sc_gemx_2025.02/analysis
Related Materials
- Obtaining full-length isoforms from single cells with nanopore sequencing whitepaper