Spatial Transcriptomics
Spatial transcriptomics enables interrogation of gene expression within the context of tissue architecture, tissue microenvironments and cell groups (especially when coupled with single cell sequencing). To meet the rapidly increasing needs for spatial -omics studies, NUSeq, along with the Pathology Core Facility (PCF), the Mouse Histology and Phenotyping Laboratory (MHPL) and the Center for Advanced Microscopy (CAM), form the Northwestern Spatial Consortium. The consortium ensures users have access to the various techniques needed to carry out a typical spatial analysis workflow, such as tissue prep, cryosectioning, staining, imaging, tissue section QC, sequencing library prep, sequencing and data analysis. The established workflow accommodates both fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues. Pre-cut tissue sections on standard glass slides may also be used for the Visium platform from 10x Genomics, as the 10x CytAssist instrument available at NUSeq enables sample transfer from pre-existing slides to Visium slides. The establishment of the spatial workflow by the consortium and the CytAssist instrument are supported by Northwestern University Office for Research.
Among the spatial platforms available, the Visium platform from 10x Genomics has been offered since February 2022. In May 2022, NUSeq received Certified Visium Service Provider status from 10x Genomics. Since September 2023, the Xenium In Situ System from 10x Genomics has been added as another new platform (see the picture and details below). Other spatial biology platforms available through the Northwestern Spatial Consortium include STOmics Stereo-seq, and Molecular Cartography from Resolve Biosciences (through a service agreement).
Xenium data from human breast cancer FFPE tissue using a 313-plex gene panel. A - Expression of selected gene markers (POSTN, IL7R, ITGAX, ACTA2, KRT15, VWF, CEACAM6, and FASN); B - H&E staining post Xenium workflow; C - Cell segmentation assigns transcripts to cells; D - t-SNE plot of different cell types in the Xenium data. (Panels A-D adapted from doi: https://doi.org/10.1101/2022.10.06.510405; Xenium image on the bottom right from 10x Genomics.)
Service Update
- Subcellular Resolution: 200 nm optical resolution, with software-enabled sub-50 nm single molecule XY localization resolution. Depth of Z-resolution is limited by tissue section thickness (10 um for FF and 5 um for FFPE);
- Sample Imageable Area: 10.45 x 22.45 mm. The system can analyze 1,400 mm2 per week;
- Tissue Compatibility: Both FF and FFPE samples;
- High Sensitivity: With the use of hybridization-based in situ sequencing (PMID: 32990747);
- High Specificity: Based on the use of barcoded padlock probes (PMID: 32990747);
- Throughput: Hundreds to thousands of genes, from the use of predesigned panels (currently including Human Breast/Brain/Lung/Multi-Tissue/Cancer Gene Expression Panels, and Mouse Brain/Tissue Atlassing Panels), predesigned panels with user add-on genes, or fully user-customized panels;
- Data Analysis: Fast on-instrument data processing including cell segmentation and clustering, post-run data analysis and visualization with Xenium Explorer and NUSeq bioinformatics support;
- Data Integration: With 10x single-cell and/or Visium spatial data;
- Allows immunofluorescence (IF), morphology (H&E) staining, or Visium spatial transcriptome analysis on the same tissue section.
Pricing by Platform and Stage
Visium (10x Genomics)
- Tissue Optimization on Test Slide (10x Visium): $750 per slide
- Sample Processing to cDNA Synthesis on Sample Slide (10x Visium): $800 per slide
- Spatial Gene Expression Library Prep from cDNA (10x Visium): $400 per slide
Xenium (10x Genomics)
- Slide Processing and Xenium Analyzer Run (up to 2 days): $4,900 for 2 slides
- Xenium Analyzer Run: $1,000 per day if additional day(s) needed
- Please check the Core Pricing webpage for external rates.
Spatial Transcriptomics
Service Request
Please contact NUSeq to initiate a spatial analysis project. The Core works closely with rest of the Northwestern Spatial Consortium on the different steps of the workflow. Project consultation is provided free of charge. Consultation with the core prior to starting a spatial transcriptome experiment is highly recommended to ensure accomplishment of project goals.
Spatial transcriptomics services can be requested through NUcore.
Sample Submission
Tissues samples that are compatible with the established 10x Genomics Visium workflow:
- Fresh frozen tissue (embedded in OCT)
- Formalin-fixed paraffin-embedded (FFPE) tissue blocks
- Fixed frozen tissue
- FFPE/fresh frozen/fixed frozen section pre-cut on regular microscopic slides
- Archived hematoxylin & eosin (H&E) stained FFPE slides
Sample RNA quality will be examined with Bioanalyzer after extraction by core staff. Only samples that pass the QC step will be accepted for spatial transcriptomic service.
Tissues samples that are compatible with the established 10x Xenium workflow:
- Fresh frozen tissue (embedded in OCT)
- Formalin-fixed paraffin-embedded (FFPE) tissue blocks
For up-to-date Tissue Preparation Guide for Visium and Xenium, please download the Demonstrated Protocol from the Visium and Xenium sites.
Bioinformatics
Data analysis is provided upon request.
Frequently Asked Questions
Visium (10x Genomics)
What is the turnaround time?
The bottleneck is typically on identifying tissues with good RNA quality. Once sample(s) pass QC and section(s) are placed on Visium slide(s), the slide(s) will be processed in 2-3 weeks and the sequencing will be performed in two weeks.
How many tissue sections can each Visium Gene Expression Slide accommodate?
For Visium slide V1 with each capture area at 6.5mm x 6.5mm, four sections can be placed on each slide. For Visium slide V2 with each capture area at 11mm x 11mm, two sections can be placed on each slide.
Can I use tissue sections that have already been prepared on regular glass slides?
Yes. The core uses the 10x CytAssist instrument to transfer tissue analytes from existing slides to Visium slides for spatial analysis.
What is the spatial resolution of the 10x Genomics Visium spatial platform?
Each spot is 55 um in diameter, providing an average resolution of 1 to 10 cells per spot. The distance from center to center between spots is 100 um. Each capture area contains approximately either 5,000 (V1) or 14,000 (V2) barcoded spots.
What is the required RNA quality for an FFPE sample to proceed to spatial transcriptomic analysis?
DV200 score needs to be >30%. DV200, i.e., the percentage of fragments >200 nucleotides, is generated from Bioanalyzer or TapeStation traces.
How does the gene expression profiling process differ between fresh frozen and FFPE tissue samples?
For fresh frozen tissues which contain mostly intact RNAs, whole transcriptomic analysis is performed through poly-T-based cDNA reverse transcription followed by sequencing. Alternatively, probe-based hybridization method can be applied as well depends on project. For FFPE samples, due to presence of RNA degradation, this is achieved through hybridization of gene-specific probes followed by sequencing.
How many genes can be detected from fresh frozen or FFPE tissue sections?
18,000 human and 21,000 mouse genes.
What is the molecular capture rate, or sensitivity, of Visium?
Molecular capture efficiency is affected by a number of factors, including tissue type, permeabilization condition, and sequencing depth. Based on publicly available Visium data from 10x Genomics, the mouse brain hippocampus region as an example can generate an average of 4,500 genes and 20,000 unique transcripts per spot, at a tissue thickness of 10 um and sequencing depth of 50,000 paired-end reads per spot.
How to calculate the number of reads needed for my samples?
The suggested sequencing depth is 50,000 and 20,000 read pairs per spot at a minimum for poly-A capture and probe-based method, respectively. The number of spots needed to cover can be estimated from the percentage of capture area covered by the tissue section. For example, if 50% of the capture area is covered based on H&E staining image, the number of read pairs needed is: 50% coverage x 5,000 total spots per capture area x 50,000 read pairs per spot = 125 million read pairs.
What sequencing length is used for Visium libraries?
Read 1: 28 cycles; Index 1 (i7): 10; Index 2 (i5): 10; Read 2: 90.
Can I integrate spatial transcriptomics data with single-cell RNA-seq data from neighboring sections?
Yes. This is achievable. Please consult the core for more specifics.
How do I start a Visium experiment?
Please contact NUSeq (NUSeq@northwestern.edu) and we will help you through the whole workflow from sample preparation to Visium slide processing to data analysis.
Xenium (10x Genomics)
How do I start a Xenium experiment?
Please contact NUSeq (NUSeq@northwestern.edu) and we will help you through the whole workflow including sample preparation, probe designation, slide processing and data analysis.
Can I use fresh frozen or FFPE samples?
Yes, both are compatible with Xenium platform.
Can I use fixed tissue embedded in OCT?
How many samples can I fit on a Xenium slide?
The imageable sample area of Xenium slide is 10.45 mm x 22.45 mm and we have to run 2 slides per run. As long as the tissue can fit into the sample area, you can place as many sample as you want.