Single Cell Scheduler

Important Information

1. This form (File:10x Single Cell Form.docx), filled out completely. Email form to the lab (ngsequencing@illinois.edu) prior to setting up your 10x experiment date.
2. Starting concentration should be at least 4,000 cells/ul (4M cells/ml) or more. We request 1M cells total, transported to us in a 1.5ml centrifuge tube. There are modified protocols for limited cells, please ask if this is your case.
3. Elimination of dead cells is critical, viability should be >70%. Your starting cell suspension will be counted upon arrival with live/dead measure included. If viability is below 70%, you may wish to repeat cell collection as background RNA levels will be high. If we are asked to continue, cells with >70% viability will be processed through the Miltenyi Biotec Dead Cell removal and counted again, which could significantly reduce total cells depending upon dead fraction.
4. Once the process above is complete, final cell suspension should have at least 20ul total volume with a concentration between 700-1,200 cells/u* (700k -1.2M/ml).
5. Each library will be made from 500-10,000 cells, depending upon your project needs. Doublets increase linearly with total cells, from ~0.6% at 1000 cells, to ~6% at 10,000 cells.

• Recommended target per sample is 3,000 cells or more. While libraries can be made to target 500-10,000 cells each, libraries capturing < 3,000 cells have much greater variability in actual cells captured (+/- 50% or more) and lower reproducibility. Variability of actual capture at or above 3,000 cells should be ~30% or less (ie: 2,700-3,300 for a 3,000-cell target). While all protocols for library preparation will be followed, due to differences in sample types submitted for 10x single cell, no guarantee is given that your cell type will behave according to typical performance.

• Prior to library setup, you will need to tell us below how many cells should be targeted for each library, and how many different samples you have.