10x Chromium Gene Expression Flex

The 10x Chromium Gene Expression Flex allows single cell human or mouse studies to be initiated from fresh tissue, fixed tissue, FACS-sorted cells, frozen tissue (nuclei) and FFPE tissue derived cell isolates. The flexibility of sample fixation and storage with fixed tissue locks the biological state of the sample while allowing for a more optimized single cell workflow including batching of fixed samples collected at divergent dates for time course studies. 10x Genomics has a preconfigure Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit, 16 rxns (1000414) which is optimized for the Gene Expression Flex protocol (inquire for KUMC contract pricing). Chromium Flex single cell expression leverages the probe based technology used for the 10x Genomics Xenium spatial expression platform for sensitive, robust performance. Chromium Flex can be perfomed with barcoded samples allowing sample plexing to reduce per sample costs and make larger-scale experiments accessible.

• Probe based single cell interrogation provides 2x greater sensitivity with Chromium Flex 
• Sample barcoding allows sample multiplexing for reduced costs
• Store fixed samples for more efficient project management
• Detect rare cell population with single cell precision.

Chromium Gene Expression Flex Unique Dual Indexed (UDI) libraries prepared using the 10x Chromium X are Illumina compatible and will be sequenced using the NovaSeq 6000 sequencer. An asymetrical sequencing profile is used to sequence the Chromium Flex single cell/nuclei libraries, therefore, only single cell Flex libraries can be pooled for a NovaSeq 6000 flow cell run. For a typical Chromium Flex expression project targeting 10,000 cells @ 15,000 reads per cell, a full S1 flow flow cell will be required to run 12 Flex libraries or 6 Flex libraries can be run on a full SP flow cell run.

Sample Submission 

The success of a Chromium Gene Expression Flex project is determine by the quality of the fixed singulated cells/nuclei provided to the Genomics Core. Researchers must coordinate their sample delivery with core personnel at least two weeks prior to submission. The cell/nuclei sample must be submitted as a fixed and completely dissassociated cell/nuclei suspension. The concentration of each cell/nuclei suspension will be assayed using the Countess II FL Automated Cell Counter.

Best Practices

• Prepare your fixed samples using the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10x Genomics 1000414).
• Plan your cell preparation time frame for the fixed samples to conform with the predetrmined Genomics Core submission schedule. Deliver singulated fixed cells/nuclei to the Genomics Core on ice. 
• Be gentle when handling your cell preparations (pipette slowly). To minimize shearing forces during pipetting use 10x Genomics validated surfactant free large bore 1000ul and 200ul pipette tips for cell manipulation and resuspension (Rainin cat#30389218 & cat#30389241).
• It is recommended that an initial fixed cell/nuclei count be performed before submitting cells to the Genomics Core to determine concentration.
• To ensure a well singulated cell/nuclei suspension free from cellular debris and cell aggregates, cell filtration can be performed using Sysmex CellTrics Filters-30uM (Fisher 04-004-2326) or MACS SmartStrainers - 30uM (Miltinyi Biotec 130-098-458). Account for volume loss associated with the filtration process and repeat the cell count to correct for lower cell counts.
• Submit samples with cell or nuclei concentration of 1000 - 2000 cells/nuclei per ul (1M - 2M cells/nuclei per ml). Higher concentrations can promote aggregation and clumping that will interfere with generating ideal single cell suspensions.
• Do not over-centrifuge cells when pelleting to obtain desired concentrations. Perform final resuspension of cells in Quencing Buffer (10x Genomics 2000516) included in Fixed RNA Sample Preparation Kit.

Supporting documents and video links for cell preparation and project planning

• Demonstrated Protocol: Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling
• Demonstrated Protocol: Fixation of Cells and Nuclei for Fixed RNA Profiling
• Single Cell Preparation Guide
• Chromium Nuclei Isolation Kit
• Tested Tissues
• Demonstrated Protocol: FFPE Tissue Dissociation
• Single Cell FFPE Tested Tissues
• Video: Chromium Single Cell Gene Expression Flex - How it Works