Feb 23, 2026
GeoMx Whole Transcriptome Atlas (WTA) Analysis of FFPE Human Term Placenta
- Scott Lindsay-Hewett1,
- Valentina Stanley1,
- Kathleen Fisch1,
- Mana Parast1,
- Louise C. Laurent1
- 1University of California San Diego
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: [email protected]
Protocol Citation: Scott Lindsay-Hewett, Valentina Stanley, Kathleen Fisch, Mana Parast, Louise C. Laurent 2026. GeoMx Whole Transcriptome Atlas (WTA) Analysis of FFPE Human Term Placenta. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk1rwwg5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 21, 2026
Last Modified: February 23, 2026
Protocol Integer ID: 243773
Keywords: GeoMx, Digital Spatial Profiler, Spatial Transcriptomics, Whole Transcriptome, Placenta, analysis of ffpe human term placenta, ffpe human term placenta, spatial transcriptomic analysis of ffpe, gene expression data from placental tissue, geomx whole transcriptome atlas, spatial transcriptomic analysis, whole transcriptome atlas, geomx digital spatial profiler, use of the geomx digital spatial profiler, resolved transcriptomic profiling, transcriptomic profiling through antibody, human term placenta, resolved gene expression data, placental tissue, ffpe
Funders Acknowledgements:
National Institutes Of Health - Female Reproductive Tissue Mapping Center
Grant ID: U54 HD104393
National Institutes Of Health - Pregnant Female Reproductive Tissue Mapping Center
Grant ID: U54 HD110347
Abstract
This protocol describes the use of the GeoMx Digital Spatial Profiler, together with the Whole Transcriptome Atlas (WTA), for spatial transcriptomic analysis of FFPE human term placenta. It outlines slide preparation, manual processing, instrument operation, and region of interest (ROI) selection, with tissue-specific parameter adjustments noted where applicable. The workflow supports compartment-resolved transcriptomic profiling through antibody-guided segmentation and is intended to enable reproducible generation of spatially resolved gene expression data from placental tissue.
Guidelines
- Ensure that all solutions are made RNase-free.
- Thoroughly treat all surfaces and Coplin jars with RNase AWAY prior to use. High-contact areas - specifically those exposed to concentrated RNA probes - must be prioritized to prevent cross-contamination in subsequent runs or downstream library preparation.
- Equilibrate deionized formamide to Room temperature prior to opening to prevent atmospheric condensation and subsequent reagent degradation. Ensure conductivity is <100 µS (use a conductivity meter). To maintain optimal stability, aliquot upon receipt and store at Room temperature .
- Buffer R contains formamide; equilibrate this reagent to Room temperature prior to opening.
- Use Lite-safe tubes when preparing antibody cocktails.
- Exercise extreme caution when applying the HybriSlip. Precise alignment is critical; an offset coverslip can cause the hybridization buffer to escape its boundaries via capillary action during the overnight hybridization, resulting in a dry slide and failed assay.
- If the HybriSlip does not detach easily after hybridization, submerge the slide in 2X SSC-T. A brief immersion (several seconds) is typically sufficient for the coverslip to slide off. Do not mechanically force the removal, as this will result in irreversible tissue damage or detachment.
- Do not allow tissue sections to dry out at any point during slide preparation.
- Ensure instrument Buffers S and H have 20% volume or more prior to starting a run.
Materials
Equipment
Microtome
Tissue floating bath
Water bath
Vortexer
Picofuge
Thermal Cycler (BioRad cat # C1000 Touch with 96–Deep Well Reaction Module); others okay
Baking oven (Amazon # B006H8JNKC)
Digital thermometer (Amazon cat # B078KPHKZD)
5.5 quart digital steamer (Hamilton Beach cat # 37530Z)
RapidFISH Slide Hybridizer (Boekel Scientific cat # 240200)
GeoMx Digital Spatial Profiler (Bruker Spatial Biology)
Materials/reagents
GeoMx DSP Instrument Buffer Kit (Bruker cat # 100474)
GeoMx DSP Collection Plate (Bruker # 100473)
GeoMx RNA Slide Prep Kit (Bruker # 121300313)
GeoMx Human Whole Transcriptome Atlas (Bruker # 121401102)
GeoMx Nuclear Stain Morphology Kit (Bruker # 121300303)
Cytokeratin, pan Antibody (AE-1/AE-3) [Alexa Fluor 532] (Novus Biologicals cat # NBP2-33200AF532)
HLA-G (4H84) Alexa Fluor 594 (Santa Cruz Biotechnology cat # sc-21799 AF594)
Alexa Fluor 647 Anti-CD31 antibody [JC/70A] (Abcam cat # ab215912)
Pipettes
Filter tips (DNase/RNase-free)
Microcentrifuge tubes (DNase/RNase-free)
Forceps (for slide handling)
Aluminum foil
Kimwipes
100% ethanol
FisherBrand Superfrost Plus microscope slides (Fisher cat # 12-550-15)
Plastic Coplin jars (Fisher cat # S89498)
StainTray Slide Staining System (Neta Scientific cat # SIM-M920-2)
HybriSlip hybridization covers (Sigma cat # GBL714022-100EA)
RNase AWAY (Fisher cat # 21-402-178)
DEPC-treated water (Fisher cat # 43-879-37)
10X phosphate buffered saline pH 7.4 (Sigma cat # P5368-10PAK)
10% neutral buffered formalin (NBF) (Fisher cat # 50-980-502)
100% deionized formamide (VWR cat # 97062-010)
20X SSC (DNase, RNase free) (Sigma cat # S6639-1L)
Proteinase K (Fisher cat # AM2546)
Antigen Retrieval Solution (Fisher cat # 00-4956-58)
Citrisolv (Fisher cat # 04-355-121)
10% Tween-20 (Fisher cat # 50-843-255)
Glycine (Sigma cat # G7126-100G)
Tris base (Sigma cat # 10708976001)
Benchtop protectors (Fisher cat # FB12007300)
Lite-safe tubes (Fisher cat # 03-391-161)
AeraSeal (Sigma cat # A9224-50EA)
Adhesive PCR foil sheets (Fisher cat # AB-0626)
Troubleshooting
Tissue preparation
1d
Using a calibrated microtome, trim the FFPE block face and section at 5 µm . Mount sections onto Fisherbrand Superfrost Plus microscope slides. Verify tissue placement is centered within the green scan area (see image below). Avoid the slide gasket (blue) or the tip calibration area (red) to prevent scanning errors.
Reproduced from the Bruker GeoMx DSP Manual Slide Preparation manual.
Tip: Print the slide template at 100% scale (actual size) to use as a physical guide for verifying correct tissue placement during mounting.
Expel residual water trapped under the paraffin section by carefully incising the wax with a razor blade and dabbing with a Kimwipe, taking care not to touch the tissue.
Air dry at Room temperature Overnight For long-term storage, slide may be kept at 4 °C or Room temperature in a sealed container with a desiccant pouch.
Manual slide preparation
2d
Slides should be processed according to the manufacturer's instructions. The GeoMx Manual Slide Preparation: RNA FFPE Quick Guide is posted here for reference.
QRG_MK4781_MAN-10130-08_GeoMx-RNA-Manual-Slide-Prep_r4_BSB-2.pdf Placenta-optimized parameters are as follows:
- Baking: 60 °C for 01:30:00 .
- Target retrieval: 100 °C in a vegetable steamer for 00:17:30 .
- Proteinase K treatment: 1 µg/mL for 00:15:00 at 37 °C .
- Hybridization: 18:00:00 overnight at 37 °C .
- Morphology marker staining: see table below
After the final 2x SSC wash, proceed to loading onto the instrument. Alternatively, slides can be stored in 2x SSC for up to 06:00:00 at Room temperature , protected from light; or in 2x SSC for up to 21 days at 4 °C , protected from light.
Instrument operation and ROI selection
1d
Slides should be loaded and scanned according to the manufacturer's instructions, and by following the onboard system prompts. The GeoMx DSP Instrument Quick Guide is posted here for reference.
QS_MK2550_MAN-10132-05_GeoMx-NGS-Instrument_r1_BSB.pdf Scan settings:
*FITC channel is selected for Auto Focus.
Following the scan, review the channel intensities. Use the software's scaling tools to manually fine-tune the display settings if the default automated levels do not provide sufficient contrast.
Initialize or assign a Readout Group, which will be the same for all collection plates in a planned sequencing run (max 8 collection plates per group).
Regions of Interest (ROIs) are selected based on morphology and antibody staining patterns. ROIs can be segmented into constituent Areas of Illumination (AOIs) using cell-type-specific segmentation rules based on antibody signal intensity. Once defined, segmentation rules apply globally to all ROIs on a given slide; however, channel thresholds can be adjusted locally to account for staining variability.
Segmentation Symbols & Rules:
When defining rules in the DSP software, each channel is assigned one of three logic gates to determine which pixels are included in an Area of Illumination (AOI):
- Plus (+): The segment includes only areas where expression is above the set threshold.
- Minus (-): The segment includes only areas where expression is below the set threshold
- Theta (ø): The channel is ignored for the purposes of generating that specific segment.
By fine-tuning these thresholds for each channel within individual ROIs, the resulting AOIs can be precisely optimized.
Placental Segment Definitions:
Note: all three segments are automatically generated for each ROI, but individual segments may be deleted if not relevant.
Villus ROIs are segmented into:
- Capillary Endothelial Cells: Defined by the "Endothelial" rule. Note: Because red blood cells (RBCs) exhibit broad-spectrum autofluorescence - particularly in the Texas Red and Cy5 channels - a Texas Red (-) requirement is imposed. This effectively "gates out" RBCs in close proximity to the capillary lining, ensuring segment purity.
- Syncytiotrophoblast: Defined by the "Trophoblast" rule.
- Villous Stroma: Defined by the "Others" rule; a "catch-all" segment, generated last, that is neither trophoblast, nor endothelial. Note: When profiling villous stroma, the segment may overlay portions of the intervillous space. This is acceptable and does not compromise data quality, as the intervillous space is acellular and will not contribute RNA to the collection.
Extravillous trophoblast (EVT) ROIs: Selected based on morphology and location, and defined by the "Trophoblast" rule. While EVT express HLA-G, the intensity varies significantly between mature (strong) and immature (weak) populations. To ensure an unbiased collection of all EVT subtypes, the HLA-G threshold is set to the maximum value (255). This effectively overrides the HLA-G (-) requirement, rendering the channel non-selective for this segment definition.
Vascular endothelial ROIs: Large vessels within stem villi are selected based on location, and defined by the "Endothelial" rule. These ROIs often require aggregation of multiple neighboring vessels to meet the minimum recommended area and nuclei count.
Advanced Parameter Tuning:
To further refine the AOIs, the following parameters are applied:
N-Dilate: Adds segment area around detected nuclei. This setting is only applicable for nuclear-tagged segments, and is not used here.
Collection & Plate Storage
3h
Upon final approval of the ROIs, initiate the automated collection sequence.
- AOI Collection: Each individual segment or standalone ROI is defined as an Area of Illumination (AOI) and is aspirated into a unique well of a 96-well collection plate.
- No Template Control: If configured during run setup, a No Template Control (NTC) will be assigned to a dedicated well. This is helpful for assessing background noise levels and potential reagent contamination during the library preparation and sequencing workflow.
- Plate Management: The system supports collection of up to 95 AOIs, plus one NTC, per plate; the software will prompt for a plate change if the collection exceeds this limit.
Post-Collection Processing
Plates must be dried before library preparation. Use one of the following methods:
- Overnight: Allow plates to dry within the instrument.
- Accelerated: Seal the plate with a breathable (permeable) membrane and dry at 65 °C in a thermocycler with the lid open for up to an hour. Check regularly and remove when DSP aspirates are dried.
Once processed, proceed to NGS library preparation and sequencing.
Storage Guidelines
If library preparation is not performed immediately, seal the plate with adhesive foil to prevent evaporation or contamination, and store according to the following intervals:
- ≤ 24 Hours: Store at 4 °C .
- 24 Hours – 30 Days: Store at -20 °C .
- > 30 Days: Store at -80 °C .
Finalizing the Readout Group
A Readout Group should be finalized once an experiment is complete or when the maximum capacity of 8 collection plates (corresponding to the 8 unique GeoMx Seq Code index plates) has been reached.
Lookup Readout Group: From the DSP Control Center, click the plate icon (bottom right), and search for the target Readout Group.
Configure NGS Readout: Enter the following parameters for a NovaSeq 6000 run:
Counting Device Model: NovaSeq 6000
Counting Platform: Illumina
Read Strategy: Paired
Read Length 1: 27
Read Length 2: 27
i5 Sequence Orientation: Reverse
Index Assignment: For each collection plate, assign one of the eight GeoMx Seq Code Index Plates (A–H). Ensure these assignments match the physical plates to be used during library preparation.
Export: Click "Finalize & Download Readout Package."
Readout Package Contents:
The downloaded ZIP folder contains three critical files required for downstream processing:
- config.ini: The configuration file required by the GeoMx NGS Pipeline to process the FASTQ files.
- LabWorksheet.txt: A metadata manifest for each AOI, documenting the specific mapping of index plates to collection plates.
- SeqCodeIndices.csv: A reference file containing the i5 and i7 index sequences necessary for sample demultiplexing.
Upon finalization, proceed to library preparation and sequencing.
Protocol references
Quick guides were obtained from the NanoString University website (https://university.nanostring.com). The latest guidance and complete manuals are also available on the site, and certain procedures and descriptions are quoted directly from the original manuals.