Does permeabilization for spatial barcode entry cause RNA leakage and affect gene expression accuracy?

Answer:
The non-ionic surfactant used in the SeekSpace spatial labeling reagent increases cell membrane permeability, enabling spatial barcodes to enter nuclei. However, its disruptive effect on the nuclear membrane is relatively limited, allowing nuclear structure to remain largely intact.

During the workflow, spatial barcodes enter nuclei and are co-captured with nuclear RNA molecules. The tissue then undergoes fixation, which stabilizes both RNA molecules and the intracellular localization of spatial barcodes, minimizing RNA leakage.

Although limited RNA diffusion may occur during permeabilization, the fixation process helps preserve nuclear RNA to maintain the accuracy of single-nucleus transcriptomic profiling and spatial expression analysis.

Key conclusions

• RNA leakage is typically minimal and has limited impact on major gene expression signals.
• High-quality samples with intact RNA and stable nuclei further reduce potential bias.
• Combined analysis and denoising of spatial barcode and RNA data help ensure reliable spatial reconstruction and gene quantification.