There are a number of reasons why the total number of identified cells is below 5,000 or above 10,000.
- The input concentration of cells is too low
The Tapestri workflow is optimized for a cell-input concentration of 300,000 – 400,000 cells per mL provided in a total volume of 35 µL. Cell input concentrations of < 300,000 cells/mL may result in lower cell throughput.
- Cell viability
Cell viability is recommended to be above 80 % as measured by Trypan Blue staining. Lower cell viability may result in reduced cell throughput due to compromised genomic DNA in individual cells or cell clumping.
- Suboptimal bead-loading
During Cell Barcoding, the current pressure profiles are programmed to target ~35 % of the encapsulated lysates to be paired with a Barcoding Bead. Decreased bead loading may lead to decreased numbers of cells. Log files from the Tapestri Instrument may be provided to Mission Bio’s support team to evaluate whether the pressure profile associated with the Barcoding Bead reservoir deviated from its target values.
- Sample loss during sample processing
Throughout the experimental workflow, samples may be lost, particularly during Breaking Emulsion and Library Cleanup.
- Input concentration of cells too high
The Tapestri workflow is optimized for a cell-input concentration of 300,000 – 400,000 cells per mL provided in a total volume of 35 µL. Cell input concentrations of > 400,000 cells/mL may result in higher cell throughput.
- Over-calling cells – Cell Finder algorithm
The model-based Cell Finder algorithm may have difficulties in automatically detecting the barcodes that are associated with high-quality cells. Please refer to the CellFinder_report.pdf document that is generated as part of the Tapestri DNA Pipeline. To resolve such an over-calling error, this may require sharing .bam files with Mission Bio’s support team.