Sep 30, 2025
Xenium Prime (5K) data generation
- xiangwei 1
- 1Washington University in St. Louis
- Washington University in St. Louis
Protocol Citation: xiangwei 2025. Xenium Prime (5K) data generation. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31717l25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 30, 2025
Last Modified: September 30, 2025
Protocol Integer ID: 228492
Keywords: data generation xenium prime, probe set of rna target, situ transcript detection, rna target, xenium prime, cell resolution, resolved expression pattern
Abstract
Xenium Prime allows for in situ transcript detection of selected probe set of RNA targets (Up to 5000 + 100) revealing spatially resolved expression patterns at single-cell resolution.
Troubleshooting
Tissue processing and QC
Determine suitable samples of interest by performing a test described in CG000578_Demonstrated_Protocol_XeniumInSituProtocolsFFPE_TissuePreparationGuide, sectioning guidelines described in Step 1 pgs. 18-25 and H&E staining in Appendix pgs. 38-45.
Evaluate section and staining quality, only choose samples or regions free of detachment or artifacts.
Xenium slide preparation
Formalin-fixed paraffin-embedded (FFPE) blocks were sectioned at 5 µm
and mounted onto Xenium slides following a modified version of the FFPE Tissue
Preparation Guide (10x Genomics, CG000578, Rev D).
Slides were deparaffinized with sequential xylene and
ethanol washes, followed by decrosslinking using the FFPE Tissue Enhancer and diluted
Perm Enzyme B (10x Genomics, CG000580, Rev E).
Xenium Prime pre-designed and add-on custom priming oligos were
hybridized to target RNAs in situ. After RNase treatment to release the RNA
strand, a polishing step was performed, followed by overnight probe
hybridization utilizing Xenium Prime pre-designed Panel
(10x Genomics) of probes for 5001 genes and an additional custom panel
of probes for extra 100 genes (10x Genomics, CG000760, Rev C).
Probes were subsequently ligated and underwent rolling circle
amplification, generating multiple copies of the gene-specific barcode for each
RNA target. Cell segmentation reagents were applied during a staining workflow
to label nuclei, membranes, and cytoplasmic regions for automated
morphology-based segmentation. The background was quenched using an
auto-fluorescence mixture, and nuclei were counterstained with DAPI to aid in
sample tracking and cell boundary segmentation (10x Genomics, CG000760, Rev C).
Xenium analyzer
Xenium slides were then loaded onto the 10X Xenium Analyzer instrument
(10x Genomics, 1000481) for imaging and initial data processing following
manufacturer’s instructions (10x Genomics, CG000584, Rev F). Fluorescently
labeled oligos bind to the amplified DNA probes, and iterative rounds of probe
hybridization, imaging, and stripping generated optical signatures for each
barcode, which were decoded to gene identities.
Post-Xenium H&E
The H&E staining of the post-Xenium slides was performed
after the run following the guidance provided (10X Genomics, CG000613, Rev B).