Imaging-based spatial transcriptomics (iST) using the Xenium
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NOTE: Do not order Xenium reagents. GECO purchases them through a blanket PO. We do not run externally purchased reagents.
Starting a 10X Genomics Xenium iST project:
Request Service: Start your Xenium iST project by requesting a service through GECO's iLab or emailing us at genome@columbia.edu (mention Xenium project in your subject line). We're happy to discuss your project with you.
Google Form: Fill out this Google Form. You will need the iLab Service ID (it has a format like this: "GECF(SB)-AB-12") to complete the form.
Pick up slides: Schedule a time to pick up Xenium slides by emailing genome@columbia.edu. When you come, bring a container with dry ice.
Prepare slides: Carefully follow the 10X Genomics Tissue Preparation Guide (see below for links). It is essential that your samples are properly fixed (or frozen), sectioned, and placed on the slide. Failure to do so can lead to run failure or sample detachment.
Return slides: Schedule a time to return your slides.
Data transfer: We will contact you when the data is ready to transfer. You must download your data within 30 days. You can analyze it using Xenium Explorer.
Xenium Protocols
Xenium Protocols
Tissue Preparation:
Xenium Data Analysis
Self-Run Analysis:
10X Genomics Analysis Tools: Analysis Guides: Xenium Explorer and Xenium Ranger
Third-party Analysis Tools: Squidpy and many other tools are linked here.
Analysis with Columbia Genome Center's Single-Cell Analysis Team:
Contact genome@columbia.edu and ask about analysis help on a Xenium project
GECO Xenium FAQ:
Why did my run fail? Why is my data quality low?
Sample Quality: How do I know if my sample quality is good enough?
How many targets do probe panels include?
How does the process for designing custom panels work?
If we purchase a custom panel, do we need to prepare all the tissue sections on slides at once?
Are there any genes that are unavailable?
How thick do samples need to be for fresh frozen and FFPE?
Which are better, fresh frozen (FF) or formalin-fixed & paraffin-embedded (FFPE) samples?
Who places the tissue sections on the slides, the user or GECO?
Do we need to fix (for fresh frozen) or deparaffinate (for FFPE) the slides ourselves?
How many sections fit on Xenium slides?
How do I transfer formalin fixed & paraffin embedded (FFPE) slides after tissue sections have been placed?
What should I do with FFPE mounted slides after placing them and fixing?
How do I store the slides after tissue placement?
What time do we need to drop off Xenium slides with samples?
Can we use the sample slides after the Xenium spatial profiling run?
How do we receive output data?
What will the output data look like?
How does the optics system work on the Xenium analyzer?
Is there a proper way to align sections on the slides?
Why did my run fail? Why is my data quality low?
Poor data quality or lost data often results from low sample quality or sample detachment from the Xenium slide. For example, both over- and under-fixation can have a negative impact on data quality. Please see the links below.
Sample Quality: How do I know if my sample quality is good enough?
We recommend that H&E staining, DAPI staining, and DV200 be performed for all samples. See this note for details.
10X Genomics claims that it has not observed a strong correlation between RIN or DV200 and run quality as long as the DV200 is at least 30 (i.e., ≥30% of RNA molecules are at least 200nt long).
10X Genomics notes that the Xenium is generally robust to the RNA degradation that is present in properly prepared and stored tissue blocks (FFPE or fresh frozen). The best quality assessment is H&E staining of a neighboring section, along with DAPI staining, as described in 10X’s sample prep protocols.
https://kb.10xgenomics.com/hc/en-us/sections/12485159296141-Tissue-and-Sample-Preparation
How many targets do probe panels include?
For pre-made panels, human tissue has a range of 266-377 genes. Mouse tissue has a range of 247-379 genes. Full lists of the genes in each panel, and their cell-type annotations, can be found on 10X’s website.
https://www.10xgenomics.com/products/xenium-panels
How does the process for designing custom panels work?
10X Genomics will work with you to choose the genes you want through their panel designer portal. Most panel design is automated and takes only a few minutes. Some panels may require advanced panel design (10X charges a fee for this).
https://www.10xgenomics.com/support/in-situ-gene-expression/documentation/steps/panel-design
https://www.10xgenomics.com/support/software/xenium-panel-designer/latest#design-inputs
If we purchase a custom panel, do we need to prepare all the tissue sections on slides at once?
No, leftover custom panel that is not used can be stored frozen. They should be used within one year following resuspension.
Are there any genes that are unavailable?
Yes. During panel design, highly expressed RNAs will be identified and dealt with using scRNA-seq data from a relevant context. There may be instances where genes that are highly and ubiquitously expressed, such as mitochondrial genes, ribosomal genes, and HLA class 1 genes are strongly discouraged on Xenium custom panels. They can take up a large portion of the available optical budget and increase the risk of optical crowding.
There may also be other (fairly rare) cases in which an RNA cannot be targeted (e.g., if the gene has very high sequence homology with another gene).
How thick do samples need to be for fresh frozen and FFPE?
10um for FF and 5um for FFPE.
Which are better, fresh frozen (FF) or formalin-fixed & paraffin-embedded (FFPE) samples?
Good results can be obtained with either FF or FFPE samples. In our experience, high-quality FF samples tend to have more transcripts per cell than high-quality FFPE samples. However, FFPE samples have much better morphology/segmentation.
Who places the tissue sections on the slides, me or GECO?
You. Or, you can work with the CUIMC molecular pathology core. You will need to pick up Xenium slides from GECO, section the tissue samples, and bring the samples back to GECO to fix or deparaffinate. Take care to follow 10X Genomics’ sample prep protocols.
Do we need to fix (for fresh frozen) or deparaffinate (for FFPE) the slides ourselves?
No, GECO will fix your frozen or deparaffinate your FFPE sample slides.
How many sections fit on Xenium slides?
The space within the fiducials on the slide is 10mm by 22mm. The Xenium can recognize 8 regions of interest (ROI) per slide. It is critical to stay within bounds of the fiducial crosshairs within the border line and not to cover any of them up or spatial profiling will not be successful.
NOTE: you can include more than 8 samples on one slide, but you will have to demultiplex them manually after the run is complete. If you want to do this, please let us know beforehand.
How do I transfer formalin fixed & paraffin embedded (FFPE) slides after tissue sections have been placed?
Bring the slides over in slide mailers at room temp immediately after the drying step in the sample prep protocol. Our sealed slide mailer is preferred, but if unavailable place the slide mailer in a secondary sealed container, such as a resealable bag.
What should I do with FFPE mounted slides after placing them and fixing?
a. Follow 10X’s sample prep protocol. Dry tissue sections upright in a drying rack at room temperature until tissue is opaque and no water remains on top of or under the section. A fan may be used to assist in drying (~10mins). If no fan is used, slides may require extended drying time for 30 mins at room temperature to ensure no water remains under the section or until dry (inspect visually, do not touch tissue).
b. Place the slides in a slide drying rack and incubate for 3 h for 42°C in a slide drying oven. (If your lab does not have an appropriate slide drying oven, please bring slides by 12pm on the day the sections were placed and we will happily do it for you.)
How do I store the slides after tissue placement?
What time do we need to drop off Xenium slides with samples?
For FF, please drop off Xenium slides with sample sections by 4pm.
For FFPE, 12pm if you need us at GECO to incubate your slides for three hours. Otherwise, if you are incubating them yourself in your own slide oven for three hours, drop them off by 4pm.
Can we use the sample slides after the Xenium spatial profiling run?
Yes, the Xenium Analyzer uses a non-destructive analysis and gentle chemistry. After the Xenium run, slides can be transferred off-instrument and stained for H&E or IF imaging. You must notify GECO in your project form if you want us to save the slides, and you must pick them up within 1 business day of the run completion.
https://www.10xgenomics.com/platforms/xenium
How do we receive output data?
The output data is about 50-100GB large, and GECO will provide a download link.
What will the output data look like?
How does the optics system work on the Xenium analyzer?
The Xenium imager uses a high numerical aperture and a fast area scan camera with a low read noise sensor to achieve ~200 nm per-pixel resolution. Depth of z-resolution is only limited by the thickness of supported tissue preservation methods (10 um for FF, 5 um for FFPE). The acquired images are processed through the Xenium Analysis software to enable single molecule detection with sub-50 nm localization resolution.
Spatial resolution: Pixel size: 0.2125 µm/pixel,
Transcript XY-localization precision < 30 nm and Z-localization precision< 100 nm
https://www.10xgenomics.com/instruments/xenium-analyzer
Is there a proper way to align sections on the slides?
As long as they are within fiducial marks, there is no proper or recommended way to align sections on the slides. Although, a uniform alignment of samples may allow for easier comparison.
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