Question: Which thermal cyclers are compatible with Xenium?
Answer: The following thermal cycler has been validated to be compatible with Xenium In Situ Gene Expression by 10x Genomics:
- Analytik Jena Biometra TAdvanced 96 SG (846-x-070-241)(where x=2 for 230 V; 4 for 115 V; 5 for 100 V, 50-60 Hz)
- Bio-Rad C1000 Touch™ Thermal Cycler with 96-Deep Well Reaction Module (Cat No. 1851197)(discontinued)
- VWR XT96 Gradient (76452-153)
- MJ Research PTC-200 (05434-05)
Thermal cyclers that are not included in the list above are unsupported as we cannot guarantee assay performance. Unsupported thermal cyclers may be incompatible with the Xenium Slide Cassette, leading to cassette leakage or evaporation, broken slides, and reduced assay sensitivity as compared to validated thermal cyclers. Cassette fit is only one component of validation; phenotypes of cassette evaporation and slide cracking are most often observed during overnight incubation and high-temperature steps.
General best practices:
1) Always place two Xenium slides in cassettes on the Xenium thermocycler adaptor.
The deep-well block allows the Xenium Thermocycler Adaptor to sit directly on top of the block for efficient heat transfer to the Xenium slide. Not using a deep-well block will lessen the heat transfer from the block to the slide and result in a decrease in assay performance.
2) Always use the Xenium Thermocycler Adaptor.
3) Ensure equipment is level.
A small bubble level can ensure the thermocycler adaptor is level for optimal assay performance. Using an instrument that is not level (as shown below) risks improper cassette sealing, evaporation, and/or uneven reagent distribution.
Please refer to thermal cycler manual for leveling instructions.
4) Pay attention to lid temperature.
The lid temperature of some thermal cyclers (Veriti, not officially supported, and XT96) do not go lower than 50°C (Veriti) or 40°C (XT96) , but some steps of the Xenium workflow calls for lower lid temperatures.
- Wash (37°C)
- Ligation (37°C)
- Amplification (30°C)
For those steps that require lower lid temperatures, we would advise turning the lid to lowest temperature possible.
'!' For the XT96, the lid will deactivate below 35°C and lead to condensation during amplification. To mitigate this, go in to System > Thermal Block > General Parameter and manually set the 'Lid Deact' field, highlighted below, to 20°C.
Alternatively, if it is not desirable to modify the XT96 general parameters, the amplification step can be set up as an incubation. It is very important if the thermal cycler is programmed as an incubation, the incubation times of 2:00:00 are followed exactly. Extending incubation times for either ligation or amplification may significantly compromise assay performance.
5) Significant evaporation can compromise assay performance.
After each thermal cycler incubation with liquid, at least 400 µl should be aspirated. If less than 400 µl of liquid is recovered, please contact 10x Support.
6) 10x recommends adjustable lid models such as the C1000.
This can help seal cassettes. Close lid and tighten until click is heard (if applicable). Do not turn past the click. If using an untested model (not recommended), models with adjustable lids are preferable.
7) Ensure thermocycler adaptor and back of slide are free of debris during incubations.
Debris in between the Xenium slide and the Xenium thermocycler adaptor can increase the risk of slide cracking on validated thermal cycler models. The risk of slides cracking with debris is higher during high-temperature steps.
For these steps, we would recommend the following best practices:
- Before placing slides on the thermocycler adaptor, wipe the thermocycler adaptor and the back of the Xenium slide with lens paper wetted with 70% ethanol or isopropanol.
- ‘!’ Avoid kimwipes as they are not lint-free.
- The thermocycler adaptor can also have debris removed with tape, but this is not recommended on the back of Xenium slides.
C1000 Best Practices:
If using thermal cyclers other than BioRad C1000, ramp rates should be adjusted for all the steps as described below:
- Analytik Jena Biometra TAdvanced: 2°C/sec heating and cooling
Product: Xenium In Situ Gene Expression