10x Genomics

FAQs

What is the minimum and maximum number of cells that can be profiled for single cell assays?

The Singl e Cell Gene Expression and VDJ assays are designed to target between 500 and 10,000 cells per sample. In cases where the sample is limiting, the following guidelines should be followed:

  • If possible, concentrate cells to achieve a cell stock concentration of ~700-1200 cells/µl
  • If concentrating the cells is not an option, load the cell suspension volume to the RT master mix without the addition of water to maximize cell count
  • In order to optimize the target cell count, extra care should be taken when assessing cell viability (>90% healthy) and cell aggregation under the microscope
  • Depending on your cell type of interest you may have very little cDNA going into the library prep. We support a wide range of total RNA and can use as little as 2 ng cDNA for library prep. If needed, the number of PCR cycles during cDNA amplification can be increased by 1-2 cycles to increase cDNA yield

For sample types with a limited number of cells, read the best practices in the Single Cell Protocols - Cell Preparation Guide ( https://support.10xgenomics.com/single-cell-gene-expression/index/doc/demonstrated-protocol-single-cell-protocols-cell-preparation-guide )

There is a 100 Cell Dataset on the 10x website that was performed with cell lines ( https://support.10xgenomics.com/single-cell/datasets/hgmm_100 )

Using more than 10,000 cells per library increases the likelihood of the sample clogging the microfluidic channels in the chip. Each chip can process eight libraries in parallel (80,000 cells per chip).

Using too many cells also increases the multiplet rate, or the chance that you have more than one cell per GEM.

What is the best way to count cells?

Accurate cell counting is critical for success with single cell experiments. The following cell counting methods are recommended:

  • Countess II Automated Cell Counter (Thermo Fisher), cell size range 7-60 µm, limited in measuring viability of very small cells
  • Hemocytometer, any cell size range
  • Scepter Handheld Automated Cell Counter (Millipore), cell size range 3-18 µm and 8-25 µm, limited dynamic range

The aim is to generate a single cell suspension between 700 - 1200 cells/µl. The cell suspension should be counted at least 3-4 times and the average of all measurements should be taken. The standard deviation between all counts should not be >25%.

A flowchart outlining the recommended sample preparation procedure can be found here

There is also a related Technical Note here

Can Cells be Sorted Prior to Running Through the 10x Single Cell Assay?

Yes, FACS samples are compatible with the 10x Single Cell workflow. It helps enrich for specific cell types based on cell surface markers and removes the dead cells and debris to produce a clean sample. If you are using a rare cell type, it is also possible to sort directly into the media that you will use with the 10x Single Cell Master Mix.

Cell counts from FACS are often overestimated, so re-count cells after flow sorting with one of the single cell counting methods listed above.

Resources

Single Cell Technology Update Webinar - Held on 13/10/20

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