![]() 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).Guided construction of single cell reference for human and mouse lung Other sensitive cell types may require washing and suspension in alternate buffers (10X validated - DPBS & HBSS). The recommended cell washing and resuspension solution is 1xPBS (calcium and magnesium free) containing 0.04% w/v BSA (400ug/ml).immortalized cell lines) and 300 rcf for 5 min. The recommended centrifugation conditions are 150 rcf for 3 min. 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 two 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 3' v3.1 expression project targeting 10,000 cells 25,000 reads per cell, a full S1 flow cell will be required to run 6 single cell 3' libraries or 3 libraries can be run on a full SP flow cell. 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. 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 expression interrogation to expose the complexity of cell diversity can have significant impact in a broad range of research areas including: ![]() 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: The latest v3.1 advancements to the Single Cell Gene Expression solution significantly improves sensitivity allowing more unique transcripts to be detected per cell. 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. ![]()
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