Spatial Collaborative Services
Spatial Collaborative Services combines expertise and technology from VAI’s Genomics Core and Optical Imaging Core to offer investigators a seamless pipeline for spatial imaging.
Additional services are in development. For questions, please email [email protected].
The Visium spatial transcriptomics platform enables whole tissue, spatially resolved, gene expression profiling in both fresh frozen and FFPE material. The system provides transcriptomic information within the context of tissue architecture, tumor microenvironments, and cell groups.
The platform relies on custom Visium slides, with 4 capture areas upon which tissue sections are affixed. Each array contains 5000 tiled capture spots with an average capture radius of 50 uM, each containing millions of oligonucleotides with a polyA capture sequence, a unique molecule identifier (UMI) and an indexed spatial barcode. After capture, Illumina-sequencing compatible libraries are generated from the probe products and used for high-throughput sequencing.
10X Visium workflow (courtesy of 10x Genomics)
The GeoMx DSP enables spatially resolved, region of interest targeted, profiling of both mRNA and proteins in both fresh frozen and FFPE tissue.
The assay is performed on standard negatively charged slides upon with the tissues of interest are mounted; antibodies or RNA probes coupled to photo-cleaveable barcode oligos are incubated with both the tissue and morphology markers. After staining, regions of interest (ROI) can be chosen and optionally subsectioned based on morphology. Oligos from the selected regions are then released by exposure to UV light, incorporated into an Illumina sequencer compatible library and used for high throughput sequencing.
Nanostring GeoMx DSP workflow (Courtesy of Nanostring)
Available Nanostring Region of Interest (ROI) segmentation options (courtesy of Nanostring)
| Platform | 10x Genomics Visium | Nanostring GeoMx DSP |
| Sample Type | Fresh Frozen or FFPE | Fresh Frozen, FFPE, Fixed Frozen |
| Submission Format | Blocks or prepared 10x custom slides | Blocks or freshly cut charged slides |
| Presubmission QC | RIN/DV200 score: Fresh = RIN >7.0; FFPE = DV200>50% | None |
| Project optimization | None | Test (slides) to assess compatibility of tissue & morphology markers |
| Imageable area | 6.5 mm x 6.5 mm. 4 regions/ one time use slide | 35 mm x 14 mm |
| Species Compatibility | Fresh Frozen = All eukaryotes; FFPE = Human/Mouse | Human/Mouse |
| RNA Compatible | Yes | Yes |
| Protein Compatible | No | Yes |
| Regions Assayed | Whole slide | Targed regions of interest, user defined |
| Resolution | ~55uM diameter (1-10 cells) | 200-600uM typical (50-100 cells) |
| Deliverables | fastq files appropriate for SpaceRanger pipeline | fastq + DSP localization files |
Frequently Asked Questions
Please fill out this form or click the button below and a core member will be in touch to schedule an initial planning meeting with the Spatial group, which includes individuals from the Genomics, Histology, Imaging, and Informatics.
Start Your Project- Embedding/re-embedding of fresh frozen/FFPE samples
- Sample quality checks (RIN/DV200 scores, H&E pathology review)
- Sectioning and section placement
- Optimization for permeabilization or antibody times
- Morphology checks (GeoMx only)
- Expression analysis
This includes imaging, slide work, library preparation, sequencing, and basic data analysis.
Questions about 10X Visium
The following samples can be used in a 10X Visium experiment:
- Fresh frozen samples of human or animal origin
- FFPE samples of either mouse or human origin
Fresh frozen samples: Flash freeze in liquid nitrogen cooled isopentane as soon as possible after collection. Optimally embed in OCT or CMC either at the same time or afterward. Tissue sections should be <6.5mm x 6.5mm to be compatible with Visium spatial slides. Optimal tissue thickness is 10um. High fat content tissues may require thicker sections.
Fresh Frozen Tissue Prep Protocol
FFPE samples: Please follow standard formalin fixation and embedding protocols. Samples should ideally be stored at 4°C post fixation to preserve RNA quality. Tissue sections should be <6.5mm x 6.5mm or <11mm x 11mm to be compatible with Visium spatial slides. Optimal tissue thickness is 5 um.
Core support is available for all QC steps listed below.
Fresh frozen:
- RNA extraction (RIN >7.0) on 10 uM section
- Visium Optimization Experiment to assess permeabilization times
Repository stored FFPE:
- RNA extraction (DV200 >50%) and H&E stain on 5 uM sections. NOTE: FFPE done on fresh samples by VAI’s Histology team do not need this step
- Visium Tissue Adhesion Test to assess optimal hydration times
- 55 um capture spots, 100um center to center. Each spot will capture 1–10 cells, depending on tissue type; each spot should not be thought of as a single cell.
- 5000 spots per 6.5mm x 6.5mm capture area.
- 14,000 spots per 11mm x 11 mm capture area.
- Entire section is captured at the same time.
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- Fresh frozen: 6.5mm x 6.5mm, 4 samples per slide
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- FFPE with CytAssist: 6.5mm x 6.5mm or 11mm x 11mm, 2 samples per slide
Question about Nanostring GeoMx DSP
The following samples can be used in a Nanostring GeoMx DSP experiment: FF or FFPE, OCT from mouse or human tissues only.
- Fresh frozen samples: Flash freeze in liquid nitrogen cooled isopentane as soon as possible after collection. Optimally embed in OCT or CMC either at the same time or afterward.
- FFPE samples: Please follow standard formalin fixation and embedding protocols. Samples should ideally be stored at 4°C post fixation to preserve RNA quality.
Optimal tissue thickness is 5um.
Tissue should be sectioned onto positively charged slides no more than two weeks prior to running the Nanostring DSP assay.
Morphology check: 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.
Minimum of 50–100 cells per ROI (region of interest). May be subdivided into multiple AOIs (areas of interest) based upon morphology markers in that region.
Multiple sections or sections from multiple tissues can be placed on the same slide, so long as the same probe set will be used on all slides.
All sections must fit within the 40mm x 17mm slide region and be entirely within the GeoMx gasket to ensure a good seal.
- Leica Bond Plus (Cat# S21.2113.A)
- Fisherbrand Superfrost Plus (Cat # 12-550-15)
- Whole transcriptome atlas (human or mouse)
- Cancer transcriptome atlas (human)
- Protein assays with NGS readout (human or mouse)