Introduction to single-cell RNA and ATAC sequencing
Single-cell and single-nucleus RNA sequencing assigns gene-expression measurements to individual cells or nuclei, while single nuclei ATAC-seq measure transposase accessible regions (markers for active gene regulatory elements) in single cells/nuclear. These assays can identify cell types, rare populations, cell states, and cell type-specific responses that maybe difficult or impossible to identify in population averaged bulk RNA-seq. Compatible additions can also measure immune-receptor clonotypes, selected cell-surface proteins, or CRISPR guide identities.
Use a cell-resolved assay when cellular heterogeneity is central to the biological question. For example, to identify rare cell populations, characterize tissue or immune cell composition, compare cell states, or determine which cell type responds to a treatment or is impacted by a disease. Single-nucleus workflows may be especially useful when intact viable cells cannot be recovered reliably from tissue.
Single-cell assays are more sensitive than bulk assays to sample quality, viability, debris, clumping, dissociation effects, ambient RNA, multiplets, and processing time. They are not necessarily the best option when only a population average is needed, when the number of biological replicates would be sacrificed to obtain more cells, or when the available material is incompatible with the selected workflow. Standard 3' and 5' gene-expression assays are gene-counting assays, not per-cell full-length isoform sequencing. Though full transcript sequencing is possible through additional library preps and Pac Bio sequencing.
Compatible sample types
Depending on the assay, projects may begin with high-quality fresh or cryopreserved cell suspensions, isolated nuclei from fresh or frozen tissue, fixed cells or nuclei, or compatible FFPE-derived material. Exact viability, concentration, volume, buffer, cell number, fixation, and storage requirements are chemistry-specific. Contact the CoLab before preparing or fixing samples.
Genomics CoLab offerings
10x Genomics 3' Gene Expression: Broad gene-expression profiling when a 5'-specific readout is not required.
10x Genomics 5' Gene Expression: Gene expression with support for immune-receptor applications such as TCR or BCR V(D)J libraries.
On-Chip Multiplexing (OCM): A cost- and batch-efficient option for compatible fresh/live 3' or 5' projects. Up to four biological samples can be combined into one downstream reaction, subject to current cell-capacity and workflow limits.
10x Flex Gene Expression: Probe-based expression profiling for compatible fixed cells, fixed tissue, nuclei, FFPE-derived material, archived samples, or highly multiplexed study designs.
Feature Barcode additions: Compatible oligo-tagged antibody, cell-surface protein, CRISPR Guide Capture, or other supported feature libraries.
TCR/BCR additions: Immune-receptor reconstruction and clonotype analysis, only compatible with 5' immune-profiling workflow.
10x Genomics single cell ATAC-seq: 10x ATAC-seq measures chromatin accessibility in individual nuclei.
10x Genomics Multiome: Measures gene expression and transposase accessibility from the same cell/nuclei.
See our single cell omics pricing.
Design considerations
When you start your project you will discuss with Genomics CoLab term the following items to assure we can meet your project needs:
- Fresh/live workflow versus fixed-sample Flex workflow.
- Intact cells versus isolated nuclei.
- 3' versus 5', including whether TCR or BCR information is required.
- Standard singleplex versus OCM for compatible multi-sample projects.
- Desired recovered cells or nuclei per biological sample and the prevalence of rare populations of interest.
- Number of biological replicates, sample synchronization, and batch balance.
- Feature Barcode, protein, CRISPR, TCR, or BCR additions and their compatibility with the selected chemistry.
- For Flex, species and probe-panel compatibility, fixation workflow, sample-barcode configuration, reactions, and cell loading.
- Whether gene expression and accessibility must be linked in the same nucleus; if not, separate assays may be simpler than Multiome.
What the CoLab performs
Depending on the selected workflow, the CoLab may review cell or nucleus quality, perform droplet partitioning and library preparation, prepare compatible add-on libraries, complete library quality control and pooling, coordinate sequencing, and provide primary processing or analysis. Tissue dissociation, nuclei isolation, fixation, and other upstream work are separate services when performed by the CoLab.
What the investigator supplies
The investigator supplies the approved cells, nuclei, tissue, or fixed material; sample metadata and experimental groups; organism information; target recovery per sample; and any investigator-provided 10x reagents, antibodies, feature reagents, or custom probes agreed during planning. Samples must be delivered according to the project-specific schedule and submission protocol.
Data and analysis delivered
Deliverables may include sequencing data, and may be expanded to include quality aligned data and QC metrics, gene-by-cell or feature-by-cell matrices, immune-receptor outputs, dimensionality-reduction and clustering results, cell annotations, marker summaries, and project-specific comparisons. Basic primary processing and more collaborative biological interpretation should be scoped separately.
Pricing and sequencing considerations
Single-cell estimates must distinguish biological samples, reactions or wells, chips, service fees, pass-through reagents, sequencing, add-on libraries, upstream sample preparation, and analysis. OCM and Flex can lower per-sample costs in suitable multi-sample designs, but their sample capacity, cell recovery, technical considerations in sample handling and preps, and molecular readouts can differ from standard singleplex. Sequencing is calculated from the target recovered cells or nuclei, recommended reads per cell for each library type, and the available sequencing formats, not from a single flat per-sample amount.
To start a project
Please download our project description form, fill it in with the as much detail as you can and then send it along with a consultation request to the [email protected] email address.