Spatial Transcriptomics
woman working in lab at computer screens

Information regarding tissue prep and sample submission for Visium, GeoMx DSP, Curio Seeker, and Xenium processing.

PlatformVisiumXeniumGeoMx DSPCurio Seeker
Sample TypeFresh Frozen, Fixed Frozen, or FFPEFresh Frozen, Fixed Frozen, or FFPEFresh Frozen, Fixed Frozen, or FFPEFresh Frozen
Submission Formatblocks, glass slides, or 10x custom slidesblocks or 10x custom slidesblocks or standard slides (freshly cut)blocks
Recommended QCFresh Frozen RIN >7; FFPE DV200 > 30%nonenoneRIN > 7
SizeFF: 6.5 mm x 6.5 mm (4 per slide)
FFPE: 6.5 mm x 6.5 mm or 11mm x 11mm (2 per slide)
10.45 mm x 22.45 mmup to 40 x 17 mm3 mm x 3mm tile; 10 mm x 10mm tile
SpeciesFresh Frozen = diverse; FFPE = human or mousediversehuman or mousediverse
AnalyteRNA and proteinpredesigned or custom targeted RNA panelsRNA or proteinRNA
Regions5000 spots per 6.5 mm capture area; 14000 spots per 11mm capture area - entire section at oncecellstargeted - user defined (shape/size) regions of interest80000 beads
Resolutionspot size 55 um diameter, avg 1-10 cells/spotsubcellularminimum 50-100 cells per ROIbead size 10 um, 1-2 cells/bead
VisualizationH&E or immunofluorescenceH&E or immunofluorescence(post Xenium processing)four-color fluorescenceserial section required
DeliverablesSpaceRanger analysis pipeline outputXenium pipeline outputQuality Control Report with R objectsCurio pipeline output
Downstream AnalysisLoupe Browser Visualization Software and 3rd party tools (e.g. Seurat)Xenium Browser Visualization Software and 3rd party tools (e.g. Seurat)vendor supplied R packagesCompatible with 3rd party tools (e.g. Seurat)
10X Genomics Visium

The Visium CytAssist workflow follows a standard histological workflow: sectioning, tissue preparation, staining (H&E or IF), and imaging all take place on a standard glass slide. Additional tissue preparation is followed by probe hybridization on the same glass slide. During this phase, probes hybridize to approximately 18,000 genes, or RNA targets, within the tissue section, offering whole transcriptome gene expression profiling. 

Next, the CytAssist instrument incorporates two normal glass slides and a Visium slide with a pair of Capture Areas. This arrangement allows for the alignment of tissue sections on the standard slides with the Capture Areas. 

The instrument uses a brightfield image for spatial referencing during data analysis, then the transcriptomic probes from the tissue hybridize onto the Visium slide. The steps that follow, starting with probe extension, adhere to the standard Visium procedure outside of the instrument.

 

 

10x Genomics Logo (Certified Service Provider)
Photo of CytAssist workflow - two normal slides + visium slides loaded into CytAssist producing alignment of tissue sections on slides with capture areas

Fresh/Frozen tissue (Manual Visium Assay)

  • Freshly obtained tissue should be snap frozen to prevent RNA degradation
  • The recommended freezing method uses an isopentane and liquid nitrogen bath
  • Frozen tissue should be embedded in Optimal Cutting Temperature (OCT), a freezing and embedding compound (can be done simultaneously with freezing process)
  • RNA quality of the tissue block should be assessed prior to sectioning. The RNA Integrity Number (RIN) should be ≥ 7
  • Each new tissue type for a lab is required to undergo a tissue optimization step so that the correct permeabilization time can be established prior to scheduling a Visium GEX appointment

CytAssist

  • Species Specific – only human and mouse samples currently supported
  • Ensure desired tissue area will fit within the 6.5 x 6.5 mm or 11 x 11 mm capture area
  • Section tissue within the allowable target area on the plain glass slide – see page 6 of the 10x Genomics recommended tissue prep guide
  • Recommended section thickness is 3-10 µm
  • RNA quality of the block should be assessed prior to sectioning. The DV200 should be ≥ 30%.

CytAssist Slide Configuration

A Visium Gene Expression (GEX) slide is comprised of two or four tissue capture areas. A standard capture area is 6.5mm x 6.5mm and contains ~5000 barcoded spots measuring 55 µm in diameter and spaced so that the distance between the centers of each spot is 100 µm. The XL capture area is 11mm x 11mm and contains ~14000 barcoded spots.


Depending on the tissue, an average of 1-10 cells will cover a spot. The spatial barcode assigned to the spot is incorporated during cDNA synthesis and enables gene expression data to be mapped back to its location within the tissue. Data is processed with the 10X SpaceRanger analysis software and can be visualized with the Loupe Browser software.

Diagram showing Visium CytAssist Spacial Gene Expression slide, Capture area, and visium gene expression barcoded spots


 

Visium Gene Expression is essentially a set of slides, reagents, and software tools that enable a person to do whole transcriptome analysis on a tissue section. Thus, there are a variety of ways you can opt to use the platform and work with the Advanced Genomics Core. 

Visium Slide Submission – Investigators section tissue onto the Visium slide. Once tissue is placed on the slide, the slide is stable at for up to four weeks so it can be easily transported on dry ice to the AGC. AGC staff will perform H&E staining, imaging, library prep, sequencing, and SpaceRanger data processing. 

Work with one of the tissue processing cores on campus – Investigators can coordinate with their preferred tissue facility to section onto the Visium slide. You can also work directly with that or another facility to stain and image the slide (required if performing IF), but you will need to meet the 10X imaging requirements. This option needs to be tightly coordinated with the AGC for hand-off of the slides. There is not a safe stopping point so AGC staff need to be prepared to accept imaged slides and proceed with the workflow.

CytAssist Workflow – Investigators section tissue onto plain glass slides. Once tissue is placed on the slide, the slide is transported to the AGC. AGC staff will perform H&E staining, imaging, library prep, sequencing, and SpaceRanger data processing. 

Tissue Submission – Tissue processed following the recommended protocols can be submitted directly to the AGC for sectioning, staining/imaging, library prep, sequencing, and data processing. The cryomold used for embedding should be of appropriate size to fit the tissue sample. Preferred size for cryoblock submission is 10 mm.

Self Service – The Visium platform does not require specialized instrumentation. Reagents can be purchased directly from 10x Genomics and users can prep samples in their own lab space. These samples are submitted to the AGC as user-made libraries ready for Next Generation Sequencing.

core stocks Visium slides

 

The core stocks Visium slides. To request a slide pick-up day, please initiate a new Spatial Transcriptomics Service Request in MiCores. This generates a service request number for your Visium project as well as a sample (slide) identification number for tracking purposes, e.g. 1234-MM-S1. Please note that the slide has a defined orientation, with the serial number information located at the top. Depending on the slide, each will contain 2-8 capture areas. Each slide will generate multiple sets of files (one per capture area) and data files for each capture area will be labeled:

  • Visium Spatial slides will generate four data sets: 1234 -MM-S1-A and 1234-MM-S1-B, 1234-MM-S1-C, and 1234-MM-S1-D.
  • CytAssist Visium slides will generate two data sets: 1234 -MM-S1-A and 1234-MM-S1-D.
  • CytAssist Visium XL slides will generate two data sets: 1234 -MM-S1-A and 1234-MM-S1-B.

*** note the first capture area is closest to the serial number at the top of the slide***

Helpful Documents

10X Genomics Xenium

FF or FFPE tissue is sectioned onto a Xenium slide (each slide has an imageable area of 12mm x 24mm). Sections are treated to access the RNA for labeling with circularizable DNA probes. Probe ligation generates a circular DNA probe which is enzymatically amplified. Slides are placed in the Xenium Analyzer where the sample undergoes successive rounds of fluorescent probe hybridization, imaging, and removal; creating bright, easy to image signal with a high signal-to-noise ratio. 

An optical signature specific to each gene is generated, enabling target gene identification. Finally, a spatial map of the transcripts is built across the entire tissue section. Data is processed with the 10x Xenium analysis software and can be visualized with the Xenium Browser software.

10x Genomics Logo (Certified Service Provider)
flow chart of 10X Genomics Xenium
  • FF or FFPE
  • Section Thickness
    • 5 µm (FFPE)
    • 10 µm (FF)
  • Tissue processed following the recommended protocols can be submitted directly to the AGC for sectioning and Xenium processing.
  • Investigators section tissue onto the Xenium slide. Once tissue is placed on the slide, the slide is stable at for up to four weeks so it can be easily transported to the AGC.

Xenium slides have an imageable area outlined by a fiducial frame measuring 12 mm x 24 mm. Specimens need to fit within the available sample positioning area measuring 235 mm2 (10.45 mm x 22.45 mm).

Xenium slides

Available now:

  • Human Brain
  • Human Breast
  • Human Colon
  • Human Lung
  • Human Multi-Tissue and Cancer
  • Human Skin
  • Mouse Brain
  • Mouse Tissue Atlas
  • Fully Custom – up to 480 genes

note – up to 100 custom targets can be added to pre-designed panels

NanoString GeoMx Digital Spatial Profiler

NanoString’s GeoMx Digital Spatial Profiler (DSP) combines spatial and molecular profiling technologies by generating digital whole transcriptome or protein expression data for up to 4 tissue slides per day. A series of imaging reagents tagged with fluorophore are used to visualize the tissue and a series of oligo-tagged profiling reagents are used to interrogate protein or transcript expression. Once regions of interest (ROIs) are selected by the investigator, DNA oligos bound to the profiling reagents are sequentially released, collected into individual wells on a 96-well plate, and subjected to Illumina library prep. Expressions levels are readout using an Illumina Sequencer and analyzed using the DSP interactive software.

flow chart of nanoString process
nanoString logo, labeled certified service provider
  • Whole Transcriptome Atlas (human or mouse)
  • Cancer Transcriptome Atals (human)
  • Protein Assays with NGS readout (human or mouse)
  • FFPE or Fresh/Frozen tissue
  • Species Specific – only human and mouse samples currently supported
  • Recommended section thickness is 5 µm
  • Tissue should be sectioned onto positively charged slides no more than 2 weeks prior to your Spatial Assay Appointment
  • A test slide for a scanning only run (without ROI definition, UV illumination, or ROI collection) is strongly recommended to expedite ROI selection the day of the experiment and validate the fluorescent staining of your morphology markers. All requests utilizing custom antibodies are required to undergo this process.

AGC GeoMx DSP Reagents June 2021 – present

Vendor Part Number Description
nanoString  GeoMx NGS RNA CTA Hs GeoMx Cancer Transcriptome Atlas
nanoString GeoMx NGS RNA WTA Hs GeoMx Human Whole Transcriptome Atlas
nanoString GeoMx NGS RNA WTA Mm GeoMx Mouse Whole Transcriptome Atlas
nanoString various Protein Module for NGS

The nanoString GeoMx DSP platform is highly interactive and projects require extensive planning. Schedule a consultation here. Once your assay has been selected, additional key decision points are:

Slides and Samples: Multiple sections or sections from multiple tissues can be placed on the same slide for DSP processing. The only caveat is that all sections must fit within the GeoMx gasket. The gasket dimensions are shown here.

Recommended slides:

  • Leica Bond Plus (Cat# S21.2113.A)
  • Fisherbrand Superfrost Plus (Cat # 12-550-15) 

Morphology Marker Selection: Morphology markers are employed to guide selection of ROIs based on visual staining patterns. While we stock nanoString morphology kits that contain common tissue markers, the DSP system is agnostic to the imaging reagents. We have a list of antibodies previously validated by the nanoString Technology Access Program. Custom antibodies are also supported as long as the antibody conjugate works with one of the 4 channels on the instrument. One of the fluorescent channels needs to be reserved for a nuclear stain, leaving three channels open for antibodies of interest. The list of available fluorescent channels are:

Channel Excitation Peak/Bandwidth Emission Peak/Bandwidth
FITC / 525 nm 480 / 28 nm 516 / 23 nm
Cy3 / 568 nm 538 / 19 nm 564 / 15 nm
Texas Red / 615 nm 588 / 19 nm 623 / 30 nm
Cy5 / 666 nm 645 / 19 nm 683 / 30 nm

Defining Regions of Interest: There are multiple approaches for defining ROIs, including placing geometric shapes around specific areas (Geometric), drawing concentric circles surrounding a histological landmark (Contour), and gridding across the entire slide (Gridded).

Regions of Interest for nanoString workflow

 

You can select 1-96 ROIs per slide as long as they do not overlap with each other. ROI size determines total counts collected. The largest allowable ROI is ~700 μm. While the smallest region for illumination is 10 μm, this is insufficient for generating profiling data. Ultimately, the minimum ROI size will depend on your tissue, the types of cells within your ROIs, and, for RNA, the transcriptional activity of the cells. We do not recommend starting with fewer than 50 cells per ROI (non-segmented). If segmenting, then larger ROIs will be required.

Regions can also be profiled using immunofluorescence/RNAscope patterns (segmentation, cell type specific). These are defined by combinations of the presence and absence of specific morphology markers will become areas of illumination (AOIs) during slide processing. Each AOI gets collected separately, thereby creating two or more expression profiles for the same ROI.

Considerations when creating segment masks include: 

  • Segments cannot overlap or extend outside an ROI 
  • Segments should be as contiguous as possible, with clean margins separating each segment from off-target tissue
  • Segments composed of numerous small particles will have reduced fidelity and higher background from adjacent tissue

Defining ROIs and their segmentation pattern is a lengthy process and requires a significant time commitment on the investigators part the day of the experiment. All first time users are required to come to NCRC for ROI selection.

Data Analysis: The AGC will complete the nanoString NGS pipeline to generate the digital count (DCC) files and notify you when they have been loaded onto the DSP. To perform data analysis, please reserve time on the instrument using our nanoString GeoMx Data Analysis calendar in MiCores. Please note days/times the instrument is available for analysis is limited based on projects the AGC is processing on the device.

Helpful Documents

Useful Links

Curio Bioscience Curio Seeker

Curio Seeker tiles are 3 x 3 mm or 10 x 10 mm and contain ~10 µm barcoded beads. The spatial barcode assigned to the bead is incorporated during cDNA synthesis and enables gene expression data to be mapped back to its location within the tissue. Data is processed with the Curio Bioscience analysis software.

flow chart of Curio Seeker User Workflow

Fresh/Frozen tissue

  • Freshly obtained tissue should be snap frozen to prevent RNA degradation
  • The recommended freezing method uses an isopentane and liquid nitrogen bath
  • Frozen tissue should be embedded in Optimal Cutting Temperature (OCT), which can be done simultaneously with freezing process
  • RNA quality of the tissue block should be assessed prior to sectioning. The RNA Integrity Number (RIN) should be ≥ 7

Curio Seeker is a set of slides, reagents, and software tools that enable a person to do whole transcriptome analysis on a tissue section. Thus, there are a variety of ways you can opt to use the platform and work with the Advanced Genomics Core. 

Tissue Submission – Tissue processed following the recommended protocols can be submitted directly to the AGC for sectioning, staining/imaging, library prep, sequencing, and data processing. The cryomold used for embedding should be of appropriate size to fit the tissue sample. Preferred size for cryoblock submission is 10 mm.

cDNA Submission – Investigators section process onto the tile, perform the Curio assay, and submits the recovered cDNA to AGC. AGC staff will perform library prep, sequencing, and Curio Bioscience pipeline data processing.

Self Service – The Curio Seeker platform does not require specialized instrumentation. Reagents can be purchased directly from Curio Bioscience and users can prep samples in their own lab space. These samples are submitted to the AGC as user-made libraries ready for Next Generation Sequencing.

Questions?
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The Advanced Genomics Core is one of the Biomedical Research Core Facilities, and a part of the Medical School Office of Research, where our mission is to foster an environment of innovation and efficiency that serves the Michigan Medicine research community and supports biomedical science from insight to impact.