Spatial Transcriptomics

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 Quality Control (QC)

All samples must pass QC before being accepted for a spatial transcriptomics service. To perform QC, core staff extracts RNA from the submitted material, and RNA quality is assessed using Bioanalyzer or TapeStation. 

Tissue Compatibility

Xenium (10x Genomics)

• Fresh frozen tissue (embedded in OCT)
• Formalin-fixed paraffin-embedded (FFPE) tissue blocks

Visium/Visium HD (10x Genomics)

• Fresh frozen tissue (embedded in OCT)
• Formalin-fixed paraffin-embedded (FFPE) tissue blocks
• Fixed frozen tissue
• FFPE, fresh frozen, or fixed frozen sections mounted on regular microscope slides 
• Archived Hematoxylin & Eosin (H&E)-stained FFPE slides

For detailed tissue preparation instructions, please see the latest demonstrated protocols for Visium/Visium HD and Xenium from the 10x Genomics site. 

Stereo-Seq (STOmics)

• Fresh frozen tissue (embedded in OCT)
• Formalin-fixed paraffin-embedded (FFPE) tissue blocks

For detailed preparation guides, please download the latest protocols for fresh frozen tissue and FFPE samples from the STOmics site. A list of validated tissues for the STOmics workflow is also available there.

Data analysis is provided upon request.

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.

Yes, both are compatible with Xenium platform.

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. 

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.

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.

Yes. The core uses the 10x CytAssist instrument to transfer tissue analytes from existing slides to Visium slides for spatial analysis.

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.

DV200 score needs to be >30%. DV200, i.e., the percentage of fragments >200 nucleotides, is generated from Bioanalyzer or TapeStation traces.

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.

18,000 human and 21,000 mouse genes.

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.

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.

Read 1: 28 cycles; Index 1 (i7): 10; Index 2 (i5): 10; Read 2: 90.

Yes. This is achievable. Please consult the core for more specifics.

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.

Stereo-seq (short for SpaTial Enhanced REsolution Omics-sequencing) is a next-generation spatial transcriptomics technology developed by STOmics. It’s designed to map the location of gene expression in tissues with ultra-high resolution and large field-of-view.

Stereo-seq uses DNA nanoball (DNB) arrays fixed on a patterned chip. Each DNB is labeled with a unique spatial barcode (known as a Coordinate ID, or CID) that corresponds to its precise location on the chip. 

1. A tissue section is placed on the chip.

2. RNA transcripts released from the tissue are captured by DNBs on the chip and reverse-transcribed into cDNA, which then carries the spatial barcode. 

3. The cDNA is made into sequencing libraries and sequenced. 

4. Bioinformatics tools then map the sequencing data back to the original spatial coordinates, creating a detailed map of gene expression across the entire tissue section.

A standard 1 cm x 1 cm Stereo-seq chip has 400 million spots.

The recommended sequencing depth for the standard Stereo-seq 1 cm x 1 cm chip for fresh frozen samples is  1.5 billion reads (~4 reads per spot). For the Stereo-seq OMNI 1 cm x 1 cm chip for FFPE samples, the recommended sequencing depth is 4 billion reads (~10 reads per spot).