Here we offer users a list of resources and protocols to help streamline the use of our core facility. We hope to standardize the methods of preparation which will optimize the potential for quality results.
- 10X User Guide
- Data Output User Guide
- Tissue Dissociation Protocol
- Human Tissue Cryopreservation Protocol
- Cell Fixation and Preservation
- Cell Hashing with FACS Staining Protocol
- CITE-seq Protocol (BioLegend)
- CITE-seq and Cell-hashing Protocol (Satija Lab)
- Hash-Staining (PostSort) Protocol
- Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols (10X Genomics)
(Must read and accept prior to use of core)
Single-cell RNA-sequencing is a cutting-edge technology that offers exciting new research opportunities. Due to the high-risk nature of the technology, we cannot guarantee successful outcome with each experiment.
Sample Preparation & Quality Disclaimer
We strongly encourage each user to optimize cell isolation and preparation as much as possible.
As a core facility, we will be happy to provide relevant information on proper isolation and enrichment techniques leading up to the use of the services offered to give the best chance at a desired outcome. We are happy to offer a “QC Check” to determine sample quality and optimization efficiency ahead of the actual experiment date.
Sample preparation and quality is of the utmost importance because unsuccessful capture is often the result of, or proportional to, the quality of the cells at input. In other cases, particular cell types fail to capture in 10X nanodroplets due to characteristics of the cell type.
If our technicians determine a poor sample quality during their own counting and QC protocols, we will notify the investigator prior to proceeding with the 10X run and make aware the heightened risk of failure due to the sample. At this point the investigator will have the choice to proceed with risk or cancel the run.
In the event of CANCELLATION, the investigator will be billed for reagents previously provided (eg. CITEseq/Cell Hashing).
In the event of PROCEEDING WITH RISK using a suboptimal sample, and a failure occurs, the investigator will be billed for reagents and labor costs associated with the steps up to the point of failure, but not for any subsequent steps following, such as sequencing or analysis.
Cell Count Submission Disclaimer
We aim to load 10,000 cells per run based on 10X Genomics protocol recommendations.
We request an absolute minimum of 30,000 live cells per sample to be submitted for each 10X run, to be confirmed by our manual counting immediately prior to processing (which is usually significantly lower than the cell counts suggested by sorters), in order to have left-over cells to re-load in the event of a machine failure.
There is the possibility of a small, but real, inherent risk of failure of the machine to produce the emulsion required to allow single cell sequencing (3% as described and documented by 10X Genomics), including but not limited to, wetting failures, pressure errors, and sample clogs.
By ensuring to provide 30,000 live cells and loading the recommended 10,000 cells, we would be able to immediately re-run the remaining left-over sample at no additional cost and avoid a completely failed experiment.
We fully understand that it is sometimes impractical to obtain 30,000 of certain rare cell populations, and we would still be willing to run samples with less than this number, but please be aware that we would not be able to re-do the 10X run in the event of a machine failure and the sample would be lost. To help mitigate the risk of machine failure resulting in the loss of irreplaceable sample, the user could consider splitting the sample in 2 parts and run them sequentially as two independent runs. However, this would result in either a smaller sample size or double the cost. In these scenarios, we advise the users to discuss the pros and cons of either approach with their PI prior to committing to running the experiment.
In the very small chance (3%) that a MACHINE FAILURE occurs while loading an optimal quality sample, the user will not be responsible for any charges.