![]() ![]() Do not over-centrifuge the cells when pelleting.Cell viabilities <70% will not be processed for single cell library preparation. 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. Submit cell samples with a concentration of 90% viability.Account for volume loss associated with the straining process and repeat the cell count to correct for cell loss. ensure that pore size is larger than the cell diameter but small enough to catch clumps and debris. To ensure a well singulated cell suspension free from cell debris and cell aggregates cell straining can be employed.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 minimizing shearing forces during pipetting use 10X Genomics validated large bore 1000ul pipette tips for cell manipulation and resuspension (Rainin cat#30389218). Be very gentle when handling your cell preparations ( pipette slowly).Solid tissues and other large cell aggregates must be disassociated using mechanical or enzymatic disassociation.Minimize delay between cell preparation and submission. Plan your cell preparation time frame to conform with the predetermined Genomics Core submission schedule.The concentration and viability of each cell suspension will be assayed using the Contess II FL Automated Cell Counter. For cell samples undergoing Flow Cytometry sorting additional coordination between the Flow Cytometry Core and the Genomics Core will be required. The cell sample must be submitted as a completely dissassociated cell suspension with a high viability percentage of >80%. Researcher must coordinate their sample delivery with core personnel at least 2 weeks prior to submission. The success of a single cell sequencing project is determined by the quality of the cell preparation provided to Genomics Core. 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. ![]() Both single cell 5' and V(D)J enriched libraries can be combined and sequenced using a 2x150 cycle sequencing profile. V(D)J enriched libraries are sequenced with a 2x150 cycle sequencing profile. An asymetrical sequencing profile is used to sequence stand alone 10X single cell 5' libraries. Single cell libraries prepared using the 10X Chromium Controller are Illumina compatable and will be sequenced using the NovaSeq 6000. 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:
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