CGI Core Services
10x Genomics Single Cell Services
Single Cell RNA-seq
The 10x Genomics Chromium is a microfluidics device capable of partitioning single cells into nanoliter emulsions for transcriptomic and genomic analyses. The CGI Core offers full-service processing of single cells into bar-coded cDNA libraries ready for sequencing on an Illumina platform. The service includes:
- Evaluation, quantification, and viability assessment of cells for input
- Single cell capture of desired target number of cells (500-20,000)
- Generation and amplification of single cell cDNAs
- Construction of Illumina sequencing-ready libraries of cDNA
- Quality assessment and quantification of libraries
Note that services do NOT include Illumina sequencing or bioinformatic analysis. Sample processing must be scheduled at least two weeks in advance. Please contact us for more information or to schedule a consultation about a project.
Spatial Transcriptomics
The 10x Genomics Visium and Visium HD platforms power the interrogation of transcription within the spatial context of intact tissues. Starting with sections from frozen, fixed frozen, or FFPE tissues, the entire transcriptome is probed or captured onto a 6.5 x 6.5mm lawn of 55 micron spots (Visium) or 2 micron squares (Visium HD) for spatial barcoding and library generation. The services include:
- H&E staining and imaging of tissue sections
- Capture of transcripts onto Visium slides using the CytAssist instrument
- Generation of Illumina sequencing-ready libraries from captured transcripts
- Quality assessment and quantification of libraries
As with single cell, services do NOT include Illumina sequencing or bioinformatic analysis. Please contact us for more information or to schedule a consultation about a project.
The core has partnered with the Markey Cancer Center Oncogenomics Shared Resource Facility to create the University of Kentucky Single Cell Researchers Network, whose goal is to serve as a community resource and user forum for investigators across campus to learn and share experiences and expertise with single cell analysis methods. To receive news and announcements from the UK scRN, join the SingleCell email listserv.
Sample Submission
At least two weeks prior to sample submission for a 10x Genomics Chromium project:
- Meet with the Imaging Center staff to discuss your project. For optimal outcomes, it is best that both the researcher and the staff understand the requirements of the project and the technology. We can also help advise you in design and sample preparation.
- Submit a completed Project Safety Questionnaire. For the safety of the staff, it is necessary to understand the nature of any hazards that may be encountered with the cells to be processed. This should be submitted at least two weeks before initiating the project, as reagents will not be purchased until the Project Safety Questionnaire has been reviewed and approved.
- Schedule a sample submission appointment two weeks in advance. Reagents for the Chromium are not stocked, so must be ordered for each run. To allow time for order processing and delivery, and to set aside staff time for sample processing, we need about two weeks' notice at this time.
- Consider a test run and evaluation of your sample prep. While not required, it is highly recommended that you schedule a trial run on your sample preparation in advance of bringing samples for 10x Chromium processing. We can evaluate your sample prep for viability, cell concentration, size, debris, and other factors that will influence the success of your run. Issues can then be identified and resolved before you bring your experimental samples for processing. There is no charge for this evaluation.
On the day of sample submission:
- Contact us when you are ready to come to the Imaging Center with your samples. If you have any changes to your schedule or if you must postpone a sample run, please contact us as soon as you know.
- Complete a Sample Submission Form and bring that with you to the Imaging Center with your samples. This may be provided either electronically or printed.
- Be prepared to stay until the initial cell assessment is performed. Potential questions or problems may arise during evaluation of the submitted cells. These may include such issues as need to change cell concentration, resolve clumping of cells, or assess sub-optimal cell viability. It is necessary to be able to consult with the researcher quickly to resolve such issues and make determinations about the best course of action. Typically, this evaluation will take about an hour, but it depends on the number of samples.
Protocols
Cell sample preparation is the most critical part of a single cell separation experiment. It is essential that the cells be:
- In single cell suspension, with no clumps or debris
- Accurately quantified, as the concentration of input cells will determine the number captured
- Highly viable (>85-90%), as dead cells will affect capture efficiency and generate noise
The preparation of high quality cells is obviously different for each type of specimen. 10x Genomics has a number of protocols suggested for some common cell and tissue types (see support link below). They can also often provide guidance for other preparations. If your tissue or cell type is not listed, contact us to help you find information to get started.
Common protocols for use with 10x Genomics Chromium services:
- 10x Genomics Cell Prep Guide
- 10x Genomics Training Video on Cell Prep
- Worthington Tissue Dissociation Guide
- 10x Genomics Protocol for Isolation of Nuclei for Single Cell RNA Sequencing
- 10x Genomics Dead Cell Removal Guide
10x Genomics provides many additional protocols, guides, and training resources at the 10x Genomics Support website.