Single-cell Omics Platform (SCOP)
The Single-Cell Omics Platform (SCOP) offers three different single-cell techniques and services, which are listed below. We provide basic bioinformatics support to annotate NGS data and the SCOP can also provide access to dissociation protocols.
- Single-cell RNA-sequencing (10x Genomics)
- Single-nucleus RNA-sequencing (10x Genomics)
- Single-nucleus ATAC-sequencing (10x Genomics; Under development)
Please take a close look at the workflow before you get in touch. The quality of the single cells/nuclei suspension is the responsibility of the user. Lumps of cells and apoptotic cells are the most common reason for a clog in the microfluidic device, which will result in an unusable library and an increased cost of the study.
We recommend that you draft a brief summary of your project, which includes the overall objective and an overview of the experimental setup. Between three and five lines is sufficient. Please note that users are charged for all reagents used for library preparation and sequencing. See more details in Pricing, below.
Before you write your summary and fill out the contact form, please read the following points in the fold-out menu below:
The preparation of single-cell suspensions is not included in the services, therefore:
- Make sure to have a validated protocol for single cell/nuclei dissociation for the target tissue. 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 we 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 send us the number of samples and techniques you would like us to run.
- Please note that our current protocols do not detect lowly expressed genes.
Kristoffer will schedule a meeting to establish feasibility, sketch experimental/analytical outline and match outcome expectations.
The amount of single-cells/nuclei captured per lane on 10X Genomics Chromium controller is dependent on the input. Please refer to the newest protocol on the 10X Genomics website. Below is our recommendation for running one lane on the 10X Genomics Chromium aiming at generating single cell transcriptome library for ~5,000 cells. SCOP will not cover the reagents if the experiments fail or do not deliver high quality data. The companies will compensate if the failure is due to manufacturing flaws of the 10X components or the Illumina system.
The three main experimental steps are:
1. 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.
2. 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 final RNA-sequencing library (Tapestation) will be performed, the concentration of library will be measure 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 RNA-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.
- Data and results handover. Formats and examples of results.
- Consideration related to data sharing upon publication (upload the GEO).
- Storage of raw data.
- User feedback
Users of SCOP's NGS 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.
The cost of single-cell RNA sequencing experiment is ~1 DKK per cell for standard projects, if fully optimized. Please see description below:
~30,000 nuclei are FACS sorted using at least 4-6 Hashtags – These cells are loaded on to a single lane on the 10X chromium controller resulting in single cell library from around 15,000 cells sequenced in a depth of ~50,000 reads per cell.
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.
We recommend running a pilot study to verify that you acquire the type of data you need before proceeding with projects of more than 15.000 cells.
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.
Please use the examples below to acknowledge SCOP in publications and remember to notify us when a SCOP acknowledgement is published.
When using data generated by SCOP and analyzed elsewhere:
- “We acknowledge The Single-Cell Omics platform at the Center for Basic Metabolic Research (CBMR) for the technical expertise and support.”
When using data generated and analyzed by SCOP:
- “We acknowledge [X], [Y], [Z] and The Single-Cell Omics platform at the Center for Basic Metabolic Research (CBMR) for technical and computational expertise and support.” (Please [insert] the initials of SCOP members that have helped you.)
When SCOP have made substantial input to the project, please consider SCOP staff as co-author(s).
Material and Method section
Contact SCOP if you need a method description of the provided NGS service(s) and please add the following acknowledgment:
“_____________ was performed by the Single-Cell Omics platform at the Center for Basic Metabolic Research.”