Single Cell Gene Expression Assays – 10X Genomic Chromium
The 10X Genomics, Chromium instrument can perform several assays from which investigators may select. There are 3’ or 5’ gene expression assays that provide data for 500 – 10,000 individual cells or nuclei per single-cell or single-nuclei suspension (scRNA-Seq or snRNA-Seq). 10X Genomics also offers scATAC-Seq (Assay for Transposase Accessible Chromatin) for use with the Chromium instrument. The scATAC-Seq assay can provide data for 500 – 10,000 individual nuclei per single-nuclei suspension.
Up to eight suspensions can be run simultaneously on one microfluidics chip for any assay selected. The system utilizes this microfluidics platform to bring together single cells or nuclei and 10x™ barcoded beads at a nanoliter scale. Once brought together, thousands of Gel Bead-In-Emulsions (GEMs) are generated where all the cDNA from a single cell share the same 10x™ barcodes.
After the initial cDNA synthesis, cDNA is amplified to generate enough product for cDNA library construction. In preparation for library construction, the cDNA is subjected to enzymatic fragmentation, size selection, end repair A-tailing, adaptor ligation and PCR. Upon completion, the library can be sequenced on an Illumina platform.
Chromium Assays
10X Genomics offers several kits to be used with the Single Cell Chromium Controller. The following are currently offered through the Single Cell Genomics Core.
Chromium Single Cell 3’ Library & Gel Bead Kit v3
- Feature Barcoding technology for Cell Surface Protein
Chromium™ Single Cell 5’ Library & Gel Bead Kit v1
- Feature Barcoding technology for Cell Surface Protein
- Chromium™ Single Cell V(D)J Enrichment Kit, Human T Cell
- Chromium™ Single Cell V(D)J Enrichment Kit, Human B Cell
- Chromium Single Cell V(D)J Enrichment Kit, Mouse T Cell
- Chromium Single Cell V(D)J Enrichment Kit, Mouse B Cell
Chromium Single Cell ATAC Library & Gel Bead Kit v1
Chromium Sample Requirements
The investigator is responsible for generating a high-quality single cell or single nuclei suspension that meets the needs of the Chromium system and the assay type selected. The following checklist has been provided to illustrate what is expected of the sample upon delivery to the Single Cell Genomics Core.
Checklist
- Concentration: Specific for assay type selected
- Volume: > 35uL preferred
- Debris-free
- Clumps (including doublets) removed
- Viability >90% preferred (unless working with nuclei or fixed cells)
- Suspension media: (should be in an approved media/buffer) Specific for assay type selected
It is best to submit a sample using optimal sample conditions to receive high quality data. If poor sample preps are provided, poor-quality data will be generated. The Core will advise on the condition of the prep upon sample submission. Lastly, practice single-cell/ single-nuclei preps are encouraged and can be brought to the Single Cell Genomics Core to be examined by a Core member upon a scheduled request.
Scheduling a Chromium Project
To schedule a 10X Genomic Chromium project please email genecore@cchmc.org.
