Single cell sequencing has become a powerful method to identify unique cell types and rare transciptomes simultaneously across a population of cells. The GEM-X Single Cell 3' Gel Beads v4 from 10x Genomics captures cells/nuclei and constructs 3' RNA-Seq libraries from single cell suspensions. Cells/nuclei are partitioned into nanoliter-scale Gel Beads-in-emulsion (GEMs) and mRNA is captured by poly(dT) primers after cell lysis. Full length cDNA is generated and tagged with 10x barcode allowing for transcripts to be associated with specific cells.
An additional primer sequence (Capture Sequence 1) enables Feature Barcode chemistry to be ran in parallel with gene expression analysis such as antibody capture of cell surface protein expression.
Key features of the service:
- Joint consultation available with Genomics Core and Bioinformatics Core.
- Cell counting with K2 Cellometer and cell suspension loading.
- Chromium X instrument to process cells with latest GEM-X reagents.
- 10x library construction and rigorous library QC.
- Sequence generation performed on a NovaSeq X Plus platform providing economical sequencing and project scalability.
- Biosafety level 2 containment available for cell collection.
Additional documentation and resources are available on the 10x Genomics support site
A meeting is required with core staff to initiate a single cell project. The meeting will discuss experimental goals as well as project turnaround times, collection scheduling, cell concentration requirements, and cell/nuclei quality requirements. Arrangements for a meeting to discuss a project can be made by sending an email to Nathan Bivens, Genomics Core director.
All projects must be initiated with a quote, and quote accepted, before a collection can be scheduled. .
The 10x Genomics Single Cell Calculator may be used to estimate the amount of sequence required for a project.
General Considerations:
- Submit cells in Eppendorf DNA Lo Bind Tubes, 2.0 ml (Cat. No. 022431048)
- Must minimize presence of cellular aggregates, dead cells, non-cellular nucleic acids and reverse transcription inhibitors.
- On the scheduled day of submission, core staff should be notified 1 hour prior to cells/nuclei being delived. It is best practice to provide periodic updates to core staff on the day of collection.
Submission Requirements:
| Cell Concentration |
Volume | Recommended buffer Buffer |
Special Instructions |
| 0.7 - 1.2 M cells/ml | 30 - 100 ul | 1x PBS with 0.04% BSA (calcium- and magnesium-free) Strongly recommended that buffer is purchased from following vendors.
|
Use wide-bore tips. Cell viability should be >70% with >90% being optimal. Filtering cell suspensions is recommended to remove debris and clumped cells. Bel-Art FlowmiTM Cell Strainer, 40 micron (Cat. No. H13680-0040) |
| Sequence Depth/Coverage* | Read Type | Index Type | Read Length | ||
| 50,000 reads per cell/nuclei | Paired end | unique dual-index | 100 bases | ||
* minimum read count recommended by MUGTC; increased sequencing depth may be desired for some studies