The NanoString GeoMx Digital Spatial Profiler spatial transcriptomics/proteomics assays are now available in partnership with the UW-Madison TRIP lab. In these assays, tissue sections are stained with probes targeting either RNA (using either the Cancer Transcriptome Atlas or Whole Transcriptome Atlas) or protein. The probes are connected to an identifier barcode oligonucleotide sequence by a UV-cleavable linker. On the GeoMx instrument, users will work with TRIP lab staff to identify regions of interest (ROIs) in their samples. These ROIs can be further subdivided into areas of illumination (AOIs). Once these areas are determined, the instrument directs UV light at each specified AOI, releasing the oligonucleotide barcodes. These barcodes are aspirated from the sample and deposited into a pre-determined well of a 96-well plate, preserving the spatial information of the AOI as these barcodes move through the assay.
Once the sample plate(s) are passed from the TRIP lab to the Gene Expression Center, we will perform an indexing PCR reaction, amplifying the material for sequencing while simultaneously applying AOI/well-specific indexes to each aspirated pool of barcodes, enabling the user to de-multiplex the libraries from the sequencing data. The DNA Sequencing core will run the raw sequencing files through NanoString’s GeoMx NGS analysis pipeline, and you will receive files that are ready to be imported into the software on the GeoMx DSP instrument for visualization and analysis with the help of a NanoString technician.
Please see the TRIP lab’s website for additional information and resources to help you plan your experiment.
NanoString GeoMx DSP Project Guidelines
Coordinate with the TRIP lab for tissue handling and submission specifics.
NanoString GeoMx GEC Submission Form
Please fill out this form and e-mail it to email@example.com prior to bringing your plate(s) and the required reagents over from the TRIP lab.
The sequencing depth for these libraries is denoted in terms of “reads per square micron” based on the total area of all the ROIs/AOIs. The simplest approach to calculating the total number of sequencing reads needed is to multiply the total area of all AOIs by the recommended reads based on the below table.
|Assay||Recommended Reads per Square Micron|
|Cancer Transcriptome Atlas (CTA)||30|
|Whole Transcriptome Atlas (WTA)||100|
|Protein||2 (per antibody used)|
Following the sample indexing PCR reaction, the standard workflow involves pooling the indexed barcodes from the entire plate into a single tube for the final bead purification steps. Per NanoString’s guidelines, you may consider requesting multiple pools depending on the nature of your experiment and your AOI selection.
Consider a hypothetical case where you choose to subdivide each of your ROIs into two AOIs: “Category A” and “Category B”, with the general observation that the “A” AOIs are much smaller in terms of AOI area (i.e. by a factor of four or more).
Single pool strategy: “Category A” and “Category B” AOIs are pooled together. Since “B” AOIs constitute a larger fraction of the pool, it is likely that the smaller “A” AOIs will be relatively underrepresented in the data. To try to avoid this, you can opt to increase the total number of sequencing reads that you purchase from the DNA Sequencing facility.
Two pool strategy: “Category A” AOIs are pooled separately from “Category B” AOIs for the final bead purification steps. Each pool’s molarity is determined separately with our final QC of the libraries. In the DNA Sequencing facility, the pools are then combined in equimolar ratios for loading into the sequencer. This strategy aims to provide more uniform sequencing coverage by normalizing the library pools to each other. This incurs small additional costs from library preparation fees, as additional reagents and consumables are needed to process and QC additional pools in parallel.
Note that these libraries are eligible for the “shared sequencing” option in the DNA Sequencing facility, meaning you purchase reads by the million. As a result, it is relatively easy to submit your libraries for additional sequencing if you start with a more conservative estimate of the number of reads that you would like to obtain.
To request a quote, please contact firstname.lastname@example.org
The cost for final library preparation for internal/UW System clients is $1,325 for one plate of up to 95 AOIs, which includes the application of the GeoMx NGS pipeline to prepare the data for analysis in the GeoMx DSP instrument. If the AOIs are are divided into multiple pools for the final steps, an additional $30 will be charged per pool. For a second plate of AOIs processed in parallel, there is an additional charge of $40 for reagents and consumables.
Please note that the sequencing costs are heavily dependent on the total area of all of the ROIs present in your plate(s) and may be further affected by additional considerations discussed in the “Guidelines” tab. As a result, it is very difficult to accurately estimate the sequencing costs for your project in advance. These libraries are eligible for the “shared sequencing” option in the DNA Sequencing facility at a cost of $6.35 per million reads for UW System users or $6.65 per million reads for external users.
As a very rough estimate, the cost of sequencing a single 300 micron diameter ROI for UW System clients is approximately $51 for the Whole Transcriptome Atlas or approximately $19 for the Cancer Transcriptome Atlas.
All data and services provided by the University of Wisconsin Biotechnology Center Gene Expression Center are intended for research purposes only. Services are not intended nor certified for diagnostic or clinical use.