10X Chromium Single Cell 3' Gene Expression Profiling
The Chromium Single Cell 3' Gene Expression solution allows the individual cell characterization and gene expression profiling of 500 to 10,000 cells per sample and 80,000+ cells per run. The latest V3 advancements to the Single Cell Gene Expression solution significantly improves sensitivity allowing more unique transcripts to be detected per cell. Combined with 10X Genomics analysis software package (Cell Ranger Analysis Pipelines and Loupe Cell Browser visualization tool) the following applications can be performed from a wide variety of cell types:
• Single Cell Sequencing
• Rare Cell Detection
• Tumor Heterogeneity
• Mechanisms of Cellular Developement
• Response to Therapeutic Interventions
• Biomarker Discovery
• Cell Atlassing
The power of Single Cell expression interrogation to expose the complexity of cell diversity can have significant impact in a broad range of research areas including:
• Developemental Biology
• Cancer Biology
• Stem Cell Biology
Single cell libraries prepared using the 10X Chromium Controller are Illumina compatable and will be sequenced using the NovaSeq 6000. An asymetrical sequencing profile is used to sequence 10X single cell libraries, therefore, only single cell libraries can be pooled for a NovaSeq flow cell run. For a typical single cell 3' v3 expression project targeting 10,000 cells @ 25,000 reads per cell, a full S1 flow cell will be required to run 6 single cell libraries.
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 prior to submission. The cell sample must be submitted as a completely dissassociated cell suspension with a high viability percentage. 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 Automated Cell Counter (ThermoFisher #AMQAX1000). a cell counting fee will be assessed for each cell sample not meeting minimum requirements or not moving forward to single cell library preparation.
• 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.
• Target cell suspensions with >90% viability. Lower cell viability will decrease the apparent efficiency of cell partitioning and recorvery 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)