Question: How do I pool custom probes for a Single Cell Gene Expression Flex + CRISPR experiment?
Answer: Similar to the guidance outlined in the Custom Probe Technical Note (CG000621), it is recommended to prepare a spike-in pool containing the RHS probes, each at a working concentration of 40 nM, and the LHS CRISPR barcoded probe at 400 nM. 5 µl of this working stock is then added to the Modified Hybridization mix. Note that these concentrations of the LHS CRISPR barcoded probe and RHS probe are the recommended starting concentrations, but additional concentration optimization may be beneficial.
Examples for preparing a Spike-In Working stock using oPools™ for RHS custom probes and Standard desalted Oligos for the LHS probes are provided below. If the spike-in pool contains more than 20 probes, this working stock may be prepared ahead of time and stored at -20C.
CRISPR probe pooling for Step 1.1 - Probe Hybridization
| Spike-in Pool Working Stock, CR001 | 1X (µl) |
| Nuclease-free water | 36 |
| LHS CRISPR, CR001 (8 µM) | 2 |
|
RHS CRISPR*, Resuspended oPool Stock (800 nM/probe) |
2 |
| Total | 40 |
*If adding probes from multiple oPools, add 2 µl from each oPool and reduce the volume of nuclease-free water proportionally.
When performing a multiplexing experiment, a stock of RHS probes may be pooled and split across multiple tubes prior to adding the LHS CRISPR barcoded probe. See example below for pooling probes for a 4-plex multiplexing experiment.
| Spike-in Pool Working Stock, CRXXX | 1X (µl) | 4X + 10% (µl) |
| Nuclease-free water | 36 | 158.4 |
| LHS CRISPR, CRXXX (8 µM) | 2 | - |
|
RHS CRISPR, Resuspended oPool Stock (800 nM/probe) |
2 | 8.8 |
| Total | 40 | 167.2 |
Add 38 µl of pooled probes to 4 individual tubes, labeled CR001 - CR004.
Add 2 µl of LHS CRISPR CRXXX to each corresponding tube.
Add 5 µl from the spike-in working stock to the Modified Probe Hybridization mix.
Additional Flex + CRISPR Resources:
- For details on how to design custom probes for CRISPR Guide Capture for use with the Flex assay please see: How do I design custom probes for CRISPR Guide Capture for use with the Single Cell Gene Expression Flex Assay?
- For details on workflow modifications required when performing CRISPR screening with the Flex assay please see: What workflow modifications are required when performing CRISPR Screening with the Single Cell Gene Expression Flex assay?
- For details on how to analyze CRISPR screening and Flex data please see: How do I analyze CRISPR screening + Single Cell Gene Expression Flex data in Cell Ranger?
While no impact on assay performance is anticipated, the use of custom probes in these assays is not officially supported by 10x Genomics. The additional guidance and resources that have been provided in this article are intended to help enable these experiments and improve customer success.
Products: Single Cell Gene Expression Flex, Fixed RNA Profiling