Question: How do I order and use custom probes in a Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) experiment?
Answer: Custom probes can be ordered from any oligo synthesis provider. In internal testing, we have tested several oligo formats available from IDT: DNA oligos (standard desalted), Ultramer™ DNA Oligos, and oPools™ Oligo Pools. All were found to have comparable results in limited testing. Please see the Technical Note Custom Probe Design for Visium Spatial Gene Expression and Chromium Single Cell Gene Expression Flex (CG000621) for additional guidance for ordering custom probes, as well as IDT's website for more information regarding the different formats.
From a convenience standpoint, ordering the custom probes as an oPool is preferred because it simplifies the pooling and dilution before use (see below). When ordering oPools™ for multiplexing experiments, RHS probes with different Probe Barcodes must be ordered as separate pools. LHS and RHS probes can be combined in the same oPools, or ordered as separate pools.
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 to improve customer success.
Pooling and dilution calculations when using custom probes
Recommended dilutions of the custom probes upstream of hybridization will depend on the ordering format. Examples for the various IDT formats - oPools™, Standard desalted and Ultramer™ DNA Oligos are provided below.
Please note that when following the pooling and dilution schemes below, all RHS probes within a pool of an intermediate stock or working stock should have the same Probe Barcode. For example, when performing a multiplex experiment using four Probe Barcodes, four spike-in pools, one for each probe Barcode, should be prepared. When adding custom probes to the hybridization reaction, the Probe Barcode for the spike-in pool must match the Probe Barcode for the WTA Probes for that hybridization reaction.
oPools™
Initial resuspension:
- Resuspend oPools™ at a final concentration of 800 nM per oligo.
- For example, for an oPools™ ordered at the 50 pmol/oligo scale, spin the tube briefly in a microcentrifuge (~5s), and then resuspend in 62.5 µl IDTE pH 8 to give a stock concentration of 800 nM/oligo. Ensure the oPools™ is completely resuspended before proceeding.
- Note this resuspension volume depends only on the oPools™ scale, it does not depend on the number of oligos in the pool. Once resuspended, oPools™ should be stored at -20℃.
Prepare a spike-in pool working stock:
- As described in the Custom Probe Technical Note, a spike-in pool containing the LHS and RHS probes, each at 40 nM, is prepared and 5 µl of this working stock is added to the Modified Hybridization mix.
Example of how to prepare a working stock solution of the spike-in pool:
| Spike-in pool working stock | Volume (µl) |
| Nuclease free water | 38 |
| Resuspended oPool | 2 |
| Final volume | 40 |
If combining probes from multiple oPools™, add 2 µl from each oPools™ and reduce the volume of nuclease free water proportionally. For example, for a spike-in working stock combining probes ordered as two oPools™, combine 2 µl of each resuspended oPools™ with 36 µl of nuclease free water into a single tube. Pipet mix 15 times with a pipet set to 30 µl. Spin down.
If combining probes from more than 20 oPools™ in a single spike-in pool, contact 10x support for additional guidance.
If the spike-in pool contains fewer than 20 total probes, it is recommended that the 40 nM per probe working stock be prepared freshly before use and then discarded. If the spike-in pool contains more than 20 probes, this working solution may be stored at -20℃.
Figure 1: Custom Probe pooling example for a 4-plex experiment using oPools™
Standard desalted or Ultramer™ DNA Oligos
When ordering probes synthesized as individual oligos in tubes or plates, the recommended dilution scheme depends on the number of probes being added in the experiment.
For <120 Total Custom Probes (<60 LHS probes + <60 RHS probes)
Initial resuspension:
- Resuspend each oligo to a final concentration of 100 µM in IDTE pH 8. Store resuspended oligos at -20℃.
Prepare an intermediate pool :
- Prepare an intermediate pool at 800 nM per probe by combining 2 µl from each 100 µM stock probe with an appropriate volume of IDTE pH 8 to give 250 µl final.
- The example below shows a spike-in pool for Probe Barcode BC001 containing a total of 6 custom probes (3 LHS + 3 RHS probes).
| Intermediate spike-in pool, BC001 | Volume (µl) |
| IDTE pH 8 | 238 |
| LHS Custom Probe 1, 100 µM stock | 2 |
| LHS Custom Probe 2, 100 µM stock | 2 |
| LHS Custom Probe 3, 100 µM stock | 2 |
| RHS Custom Probe 1, BC001, 100 µM stock | 2 |
| RHS Custom Probe 2, BC001, 100 µM stock | 2 |
| RHS Custom Probe 3, BC001, 100 µM stock | 2 |
| Final volume | 250 |
- Vortex 30 sec and spin down. This intermediate pool may be stored at -20℃.
- Prepare a spike-in pool working stock
- As described in the Custom Probe Technical Note, a spike-in pool containing the LHS and RHS probes, each at 40 nM, is prepared and 5 µl of this working stock is added to the Modified Hybridization mix.
- Pipet mix 15 times with a pipet set to 30 µl. Spin down.
Example of how to prepare a working stock solution of the spike-in pool.
| Spike-in pool working stock | Volume (µl) |
|---|---|
| Nuclease free water | 38 |
| Intermediate pool, 800 nM/probe | 2 |
| Final volume | 40 |
If the spike-in pool contains fewer than 20 total probes, it is recommended that the 40 nM per probe working stock be prepared freshly before use and then discarded. If the spike-in pool contains more than 20 probes, this working solution may be stored at -20℃.
Figure 2: Custom Probe pooling example for 4-plex experiment using standard desalted oligos
For < 2500 Total Custom Probes (<1250 LHS probes + <1250 RHS probes)
Initial resuspension
- Resuspend each oligo to a final concentration of 100 µM in IDTE pH 8. Store resuspended oligos at -20℃.
- Prepare a spike-in pool working stock
- As described in the Custom Probe Technical Note, a spike-in pool containing the LHS and RHS probes, each at 40 nM, is prepared and 5 µl of this working stock is added to the Modified Hybridization mix.
- Combine 2 µl from each 100 µM stock probe with an appropriate volume of nuclease free water to give 5 mL final. The example below shows a spike-in pool for Probe Barcode BC001 containing a total of 300 custom probes (150 LHS + 150 RHS probes).
-
Vortex 30 sec and spin down. The spike-in working solution can be stored at -20℃ in aliquots to minimize repeated freeze-thaw.
| Spike-in pool working stock, BC001 | Volume (µl) |
| Nuclease free water | 4400 |
| LHS Custom Probe 1 through Custom Probe 150, 100 µM stock | 2 µl each |
| RHS Custom Probe 1 through Custom Probe 150, BC001, 100 µM stock | 2 µl each |
| Final volume | 5000 |
For > 2500 Total Custom Probes
Please contact 10x Support for additional guidance.
Products: Single Cell Gene Expression Flex, Fixed RNA Profiling