The Single 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.
