Question: How do I design custom probes to detect fluorescent reporters EGFP and/or RFP for use with Visium Spatial Gene Expression for FFPE, Visium CytAssist Spatial Gene Expression, or Chromium Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) assay?
Answer: To detect fluorescent EGFP and/or RFP fluorescent reporters, probe pairs were designed against common forms of EGFP and RFP sequences using reference sequences from Addgene (EGFP, https://www.addgene.org/13031/, RFP https://www.addgene.org/13032/). We strongly recommend verifying that the target sequence contained within these probe designs is the reverse complement of the EGFP or RFP sequence being targeted in a given experiment to determine if these will be compatible.
The LHS probes are listed in Table 1 and the RHS probes for Flex (i.e. Fixed RNA Profiling) are listed in Table 2, while the RHS probes for Visium Spatial Gene Expression for FFPE or Visium CytAssist Spatial Gene Expression are listed in Table 3.
Table 1. LHS probe sequences for detection of EGFP and RFP reporter genes
| Custom LHS Probes, compatible with Flex or Visum for FFPE | |
| Probe name | 5’-CCTTGGCACCCGAGAATTCCA-target_LHS-3’ |
| EGFP-LHS-1 | CCTTGGCACCCGAGAATTCCAggtagtggtcggcgagctgcacgct |
| EGFP-LHS-2 | CCTTGGCACCCGAGAATTCCAagggtgtcgccctcgaacttcacct |
| EGFP-LHS-3 | CCTTGGCACCCGAGAATTCCAatggtgcgctcctggacgtagcctt |
| RFP-LHS-1 | CCTTGGCACCCGAGAATTCCAtcggtcttgtaggcgccgggcagct |
| RFP-LHS-2 | CCTTGGCACCCGAGAATTCCAaagttggtgccgcgcagcttcacct |
| RFP-LHS-3 | CCTTGGCACCCGAGAATTCCAcctcgatctcgaactcgtggccgtt |
LHS probe designs may be used with RHS probes for Flex, containing any Probe Barcode, BC001-BC016 (examples listed in Table 2) or with RHS probes for Visium Spatial Gene Expression for FFPE or Visium CytAssist Spatial Gene Expression (listed in Table 3).
Table 2. RHS probe sequences for detection of EGFP and RFP reporter genes for Chromium Single Cell Gene Expression Flex, shown with Probe Barcode BC001.
| Chromium Single Cell Gene Expression Flex Custom RHS Probes | |
| Probe name |
/5Phos/-target_RHS-ACGCGGTTAGCACGTA-NN-ACTTTAGG-CGGTCCTAGCAA-3’ Constant Sequence Probe Barcode |
| EGFP-RHS-1 | /5Phos/gccgtcctcgatgttgtggcggatcACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA |
| EGFP-RHS-2 | /5Phos/cggcgcgggtcttgtagttgccgtcACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA |
| EGFP-RHS-3 | /5Phos/cgggcatggcggacttgaagaagtcACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA |
| RFP-RHS-1 | /5Phos/gcacgggcttcttggccatgtaggtACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA |
| RFP-RHS-2 | /5Phos/tgtagatgaactcgccgtcctgcagACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA |
| RFP-RHS-3 | /5Phos/cacggagccctccatgcgcaccttgACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA |
RHS probe sequences for detecting EGFP and RFP shown in Table 2 are for use in a singleplex Flex experiment containing Probe Barcode BC001. For multiplexed experiments, please follow the guidance outlined in How do I design custom probes for a Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) for multiplexed samples experiment?
Table 3. RHS probe sequences for detection of EGFP and RFP reporter genes for Visium Spatial Gene Expression for FFPE
| Visium Spatial Gene Expression for FFPE Custom RHS probes | |
| Probe name | /5Phos/-target_RHS-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3’ |
| EGFP-RHS-1 | /5Phos/gccgtcctcgatgttgtggcggatcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA |
| EGFP-RHS-2 | /5Phos/cggcgcgggtcttgtagttgccgtcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA |
| EGFP-RHS-3 | /5Phos/cgggcatggcggacttgaagaagtcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA |
| RFP-RHS-1 | /5Phos/gcacgggcttcttggccatgtaggtAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA |
| RFP-RHS-2 | /5Phos/tgtagatgaactcgccgtcctgcagAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA |
| RFP-RHS-3 | /5Phos/cacggagccctccatgcgcaccttgAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA |
RHS probe sequences for detecting EGFP and RFP shown in Table 3 are for use in either a Visium Spatial Gene Expression for FFPE or Visium CytAssist Spatial Gene Expression experiment. Note, the LHS probes listed in Table 1 are to be used with the corresponding RHS probes listed here.
Shown below are examples of EGFP and RFP detection in Chromium Single Cell Gene Expression Flex singleplex (Figure 1) and multiplex (Figure 2) experiments, using the EGFP and RFP custom probe designs listed in Tables 1 and 2.
Figure 1: Detection of EGFP and RFP in a Singleplex Flex experiment using custom probes
A mixture of custom probes detecting EGFP and RFP were added to individual hybridization reactions containing EGFP- or RFP-expressing cells (A). EGFP is detected only in cells expressing GFP but is not detected in cells expressing RFP. Similarly, RFP is only detected in those cells expressing the RFP reporter but not in GFP expressing cells (B).
Figure 2: Detection of EGFP and RFP in a Multiplex Flex experiment using custom probes
A 4-sample multiplex experiment was performed in which a mixture of custom probes detecting EGFP and RFP were added to individual hybridization reactions containing EGFP, RFP, a 50:50 mixture of EGFP:RFP cells or negative control cells (A). After hybridization, samples were pooled in equal proportion, washed, and loaded into a single GEM lane. Using a UMI threshold of 1 or greater, EGFP is detected only in samples containing EGFP cells (BC001 and BC003) but is not detected in samples expressing RFP only (BC002) or in negative control cells (BC004). Similarly, RFP is only detected in samples expressing the RFP reporter (BC002 and BC003) but not in the EGFP-only sample or in the negative control (B), with a UMI cutoff of 1 or greater.
While no impact on assay performance is anticipated, the use of custom probes in these assays is not officially supported by 10x Genomics. 10x Genomics cannot guarantee that custom probes will yield data comparable to that from the whole transcriptome panel. 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, Visium for FFPE, Visium CytAssist for FFPE