Question: Why do I need to calculate cDNA length for the Spatial Targeted Gene Expression assay?
Answer: When using Visium Spatial Gene Expression libraries as input into the Targeted Gene Expression assay, it is important to follow the appropriate protocol based on the average fragment length of the cDNA that was used to generate the parent Visium Spatial Gene Expression library.
When generating cDNA in the Visium Spatial Gene Expression workflow, the average fragment length of the cDNA can vary based on the tissue type and tissue quality. Some tissue types have high levels of RNases, which can degrade RNA and lead to short cDNA. Furthermore, delays during tissue processing and suboptimal tissue storage conditions can also cause RNA degradation, which can lead to short cDNA.
When short cDNA is used to generate Visium Spatial Gene Expression libraries that are used as input into the Targeted Gene Expression assay, this leads to library fragments that have a shorter baited transcript length. To ensure that these library fragments are captured efficiently by baits in the Targeted Gene Expression assay, the following workflow modifications are necessary:
- Increasing the hybridization time from 2 hours to overnight, to increase the capture rate
- Decreasing the capture and wash temperature from 65 °C to 60 °C to decrease the wash stringency
Product: Targeted Gene Expression, Visium for fresh-frozen