# Single Cell Fixed RNA Profiling Probe Sets

The Single Cell Fixed RNA Profiling assay uses probes that target protein-coding genes in the human or mouse transcriptome. Each probe consists of a pair of oligonucleotides that hybridize to the targeted transcript and are subsequently ligated.

Gene IDs and probe sequences are defined in the probe set reference CSV input file for cellranger multi. Three supporting files are provided: a BED file listing the reference genome coordinates for each probe, a CSV file listing probes with predicted off-target activity that are excluded from analysis by default, and a metadata TSV file that contains gene name and description for each of the probes. These files are described in detail on the Probe Sets Description page.

1. What genes are excluded from the human and mouse probe sets?

Below are the genes excluded:

• TCR joining and variable regions
• IG joining and variable regions
• Ribosomal proteins
• Mitochondrial ribosomal proteins
• KIR and HLA genes (due to their inherent allelic diversity)
• Non-coding RNA

2. Which genes have 1-fold coverage versus 3-fold coverage in the probe set?

Most genes have 3-fold coverage. The genes with 1-fold coverage include highly expressed genes and mitochondrial protein coding genes. This design dampens the reads on highest expression genes. A very small fraction of genes with specificity issues during probe design have 1- to 2-fold coverage.

• Human probe set: 7.5% genes have 1-fold coverage. About 5% of genes have more than three probes to cover all possible isoforms.
• Mouse probe set: 6.6% genes have 1-fold coverage. 1.7% of genes have more than three probes to cover all possible isoforms.

The information on the coverage for genes can be found in the probe set metadata TSV file.

3. Are these probes expected to work equally well on pre-spliced mRNA from nuclei?

Most of the probes do not overlap splice junctions, so they should work well with pre-spliced mRNA from nuclei.

• Human probe set: 14% (7,540) of 54k probes span a splice junction by at least 10 bp and would be less likely to detect pre-mRNA.
• Mouse probe set: 16% (8,714) of 55k probes span a splice junction by at least 10 bp and would be less likely to detect pre-mRNA.

Users can find out whether a probe spans a splice junction by looking at the probe set BED file. If the value in the 10th column (blockCount) is more than 1, it means the probe spans a splice junction.

In v1.0.1 probe sets (compatible with Cell Ranger v7.1), the region column of the probe set reference CSV file also indicates whether a probe spans a splice junction by at least 10bp (spliced) or not (unspliced). Note that most genes have multiple probes (3-fold coverage, see question 2), and not all probes for one gene will cover the splice junction.

4. Would the human probe sequences work on non-human primate samples?

The human probe sets are designed based on the GENCODE basic transcript annotations for human and validated with human samples. We have not tested the human probe set with samples from other species (including primates), and the performance of the human probe set cannot be guaranteed for other species.

5. Can custom probes be used with the Chromium Single Cell Gene Expression Flex assay?

Refer to the Custom Probe Design for Visium Spatial Gene Expression and Chromium Single Cell Gene Expression Flex Technical Note for guidance.