10x Chromium Single Cell 5' and V(D)J Enrichment
Andrew Patterson The Chromium Single Cell 5' Gene Expression solution allows the individual cell characterization of 500 to 10,000 cells per sample and 80,000+ cells per run. Combined with the optional V(D)J enrichment for immune profiling of human or mouse T or B cells, a comprehensive cellular context of the adaptive immune response and immune repertoires of ≤80,000 human or mouse T or B cells can be determined on a cell-by-cell basis per 10x Chromium run. The 10x Genomics workflow allows your project to reveal insights into T and B cell diversity, V(D)J recombination and immune cell profiling.
- Reveal clonality, diversity, antigen specificity and cellular context
- Assemble and annotate full length V(D)J gene sequences
- Pair α and β chain TCR sequences from individual T cells
- Pair heavy and light chain immunoglobulin (Ig) sequences from individual B cells for full isotype resolution
- Simultaneously measure TCR, B cell Ig, cell surface protein expression and 5' gene expression in the same cells
- Link full length, paired TCR α and β chain sequences with TCR-pMHC specificity
- Simultaneously measure cell surface protein expression with gene expression (using the Feature Barcoding application - inquire)
The power of Single Cell 5' expression combined with V(D)J enrichment immune profiling can provide significant understanding of the adaptive immune system in the following research areas:
- Immunology
- Neurology
- Stem Cell Biology
- Immuno-oncology
- Infectious disease
Single cell libraries prepared using the 10x Chromium X are Illumina compatable and will be sequenced using the NovaSeq 6000. An asymetrical sequencing profile is used to sequence stand alone 10x single cell 5' libraries. V(D)J enriched libraries are sequenced with a 2x150 cycle sequencing profile. Both single cell 5' and V(D)J enriched libraries can be combined and sequenced using a 2x150 cycle sequencing profile. For a typical single cell 5' expression project with V(D)J enrichment targeting 10,000 cells @ 25,000 read pairs per cell for 5' libraries and 5000 read pairs for V(D)J enriched libraries, a full S1 flow cell will be required to run 6 combined libraries or 3 combined libraries on a full SP flow cell.
Sample Submission
The success of a single cell sequencing project is determined by the quality of the cell preparation provided to Genomics Core. Researcher must coordinate their sample delivery with core personnel at least 2 weeks prior to submission. The cell sample must be submitted as a completely dissassociated cell suspension with a high viability percentage of >80% . For cell samples undergoing Flow Cytometry sorting additional coordination between the Flow Cytometry Core and the Genomics Core will be required. The concentration and viability of each cell suspension will be assayed using the Contess II FL Automated Cell Counter.
Best Practices
- Plan your cell preparation time frame to conform with the predetermined Genomics Core submission schedule. Minimize delay between cell preparation and submission. Keep cells on ice.
- Solid tissues and other large cell aggregates must be disassociated using mechanical or enzymatic disassociation.
- Be very gentle when handling your cell preparations (pipette slowly). To minimizing shearing forces during pipetting use 10x Genomics validated large bore 1000ul pipette tips for cell manipulation and resuspension (Rainin cat#30389218).
- It is recommended that an initial cell count be performed before submitting cells to the Genomics Core to determine cell concentration and cell viability.
- To ensure a well singulated cell suspension free from cell debris and cell aggregates cell straining can be employed. ensure that pore size is larger than the cell diameter but small enough to catch clumps and debris. Account for volume loss associated with the straining process and repeat the cell count to correct for cell loss .
- Submit cell samples with a concentration of <5000 cells/ul (5 million cells/ml). Maintaining cells at higher concentrations can cause aggregation and clumping that will interfere with generating ideal single cell suspensions. Target cell concentrations between 1000 - 5000 cells/ul (1M - 5M cells/ml).
- Target cell suspensions with >90% viability. Lower cell viability will decrease the apparent efficiency of cell partitioning and recovery since non-viable and dying cells generally contain less RNA which is more fragmented. Cell viabilities <70% will not be processed for single cell library preparation.
- Do not over-centrifuge the cells when pelleting. The recommended centrifugation conditions are 150 rcf for 3 min. at RT° for larger cells (ie. immortalized cell lines) and 300 rcf for 5 min. at RT° for smaller cells (ie. PBMCs).
- The recommended cell washing and resuspension solution is 1xPBS (calcium and magnesium free) containing 0.04% w/v BSA (400ug/ml). Other sensitive cell types may require washing and suspension in alternate buffers (10x validated - DPBS & HBSS). If viability cannot be maintained in one of these buffers, 10x has verified the following media to be compatible with 10X single cell protocols (EMEM+10%FBS, DMEM+10%FBS, IMEM+10%FBS, RPMI+10%FBS, Ham's F12+10%FBS, 1:1 DMEM/F12+10%FBS, M199).
