Single-cell Omics Platform (SCOP)
The Single-Cell Omics Platform (SCOP) offers different single-cell services, which are listed below. SCOP provides basic bioinformatic support to annotate sequencing data and we can also provide access to dissociation protocols.
3’RNAseq v. 3.1 (10x Genomics) is a high through-put transcriptomic analysis with detection of up to 2000-3000 transcripts per cell.
3’RNAseq (10X genomics) with antibody-based cell surface or nuclei detection. Using cell hashing, samples can be multiplexed across several 10X reactions whereby technical viability will be reduced.
ATAC-seq v. 1.1 (10X Genomics) at a single cell resolution for the analysis of chromatin accessibility.
Multiome, single-nucleus RNA and ATAC sequencing
- RNA and ATAC sequencing from the same nucleus (10X Genomics).
Single-nucleus Cut & Tag
- The mapping of histone marks associated with accessible chromatin in a single cell resolution.
Please read though our guidelines below.
The preparation of single-cell suspensions is not included in SCOP’s services therefore:
Make sure to have a validated protocol for single cell/nuclei dissociation for the target tissue as the quality of the single cells/nuclei suspension is the responsibility of the user. Contact the platform to consult on the dissociation protocol.
Good quality of cells/nuclei are essential for good data, so we recommend that you perform a pilot study to verify that your specific single cell/nuclei preparation will generate good data – we do not cover the cost of failed samples.
The platform provides only basic bioinformatics analyses:
Make sure that you can analyze normalized gene expression yourself. The platform ‘only’ provides basic bioinformatics analyses (UMAP cluster plots, marker gene lists, differentially expressed genes across experimental conditions).
User fees apply:
Make sure to have the funding to cover consumables. For example, one sample (~10.000 cells/nuclei ~20-40µl) will result in ~5,000 single cells/nuclei and costs ~DKK 16,000 including RNA-sequencing.
Note that SCOP cannot cover the cost in the case of failure. If the failure is due to manufacturing flaws of the 10X components or the Illumina system, the companies will compensate.
If you would like us to provide an early cost estimate, then please contact us (LINK: CBMR-SCOP@sund.ku.dk) with information about the number of samples and techniques you would like us to run.
Please note that our current protocols do not detect lowly expressed genes.
The three main experimental steps are:
1. Cell hashing
Cell hashing is the responsibility of the user and samples should be incubated with cell hashing antibodies (Hashtags) prior sample drop-off. Contact SCOP to consult on cell hashing, and to receive a cell hashing protocol and Hashtags (SCOP offers only Hashtags for nuclei).
2. Sample drop-off
Single cells or nuclei should be delivered in a concentration ~250-500 dependent upon the desired amount of cells that need to be analysed, which will be decided upon in the project planing. The quality of the cells/nuclei will reflect the downstream process – poor quality leads to poor data, we do not perform a control for amount or quality of the cells/nuclei.
3. Single cell library preparation
The cells will be loaded on the 10X Genomics Chromium and processed according to standard protocol.
- QC of cDNA (Tapestation) will be performed, and part of the cDNA will be saved for alternative processing.
- QC of final RNA-sequencing library (Tapestation) will be performed, the concentration of library will be measured to determine the ratio for pooling prior to RNA-sequencing.
Relevant samples will be pooled and sequenced on an Illumina NovaSeq aiming for a theoretical number of 50,000 reads per cell (the amount of reads per cell can be adjusted in communication with the user). Normally there will be excess of library that will be stored for potential additional re-sequencing, if requested by the user. All solutions (leftover cDNA and library) has to be picked up by the user within one month of sequencing.
The data processing and analysis may include any combination of the following steps:
- Normalisation of single cell next-generation sequencing data.
- Construction of single cell RNA-seq cell type cluster plots.
- Data-driven identification of cell type marker genes. The user can specify a number of markers for each cluster.
- Alignment with other single cell RNA-seq data sets.
Identification of cell type-specific differentially expressed genes across conditions.
Users of SCOP's single-cell services are charged for the reagents used for library preparation and sequencing. SCOP covers all expenses associated with work time and currently SCOP is offering a 30% discount on the total price for all CBMR users. Collaborators cover 100% of the reagent cost.
The cost of single-cell RNA sequencing experiment is ~1 DKK per cell for standard projects, if fully optimized.
Above price is 1.25 DKK per cell. To hit the magic price of ~1 DKK per cell you need to analyze ~70,000 cells as the sequencing gets cheaper per cell with bigger sequencing kits.
Please recognize and acknowledge SCOP’s contributions to the scientific improvements in your project. Being acknowledged in publications is very important as it serves as documentation for the SCOPS's value and performance.