May 28, 2026
Spatial RNA and Protein Analysis of FFPE Placental Tissue Using the Singular Genomics G4X Platform
- Braulio Banuelos1,
- Gina Benedetto1,
- Sabrina Shore1,
- Scott Lindsay-Hewett2,
- Morgan Meads2,
- Valentina Stanley2,
- Mana Parast2,
- Louise Laurent2
- 1Singular Genomics Systems, Inc;
- 2University of California San Diego
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: [email protected]
Protocol Citation: Braulio Banuelos, Gina Benedetto, Sabrina Shore, Scott Lindsay-Hewett, Morgan Meads, Valentina Stanley, Mana Parast, Louise Laurent 2026. Spatial RNA and Protein Analysis of FFPE Placental Tissue Using the Singular Genomics G4X Platform. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvww3m9vmk/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: May 07, 2026
Last Modified: June 02, 2026
Protocol Integer ID: 316538
Keywords: protein analysis of ffpe placental tissue, ffpe placental tissue, multiplex characterization of placental tissue architecture, placental tissue architecture, placental tissue section, singular genomics g4x platform, plex custom placenta transcript panel, singular genomics g4x platform this protocol, ffpe tissue block, g4x flow cell, spatial rna, spatial detection of rna, spatial multiomic analysis of formalin, protein analysis, plex immuno, spatial multiomic analysis, rna, protein target
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 preparation and spatial multiomic analysis of formalin-fixed paraffin-embedded (FFPE) placental tissue sections using the Singular Genomics G4X Platform. FFPE tissue blocks are serially sectioned and mounted onto slides for downstream processing. Slides undergo deparaffinization, tissue preparation, and probe hybridization using a targeted 306-plex custom placenta transcript panel together with a standard 16-plex immuno-oncology (IO) antibody panel. Following assay preparation, slides are assembled into G4X flow cells and loaded onto the instrument for automated imaging and spatial detection of RNA and protein targets. The resulting datasets enable multiplex characterization of placental tissue architecture, cellular composition, and molecular expression patterns.
Materials
See the attached G4X Sample Preparation Guide.
Tissue collection and preservation
Placenta biopsies were collected as previously described, except that the full tissue thickness was preserved in FFPE, rather than separating fetal and maternal portions, to take advantage of the large G4X scan area (5 × 1 cm per lane in the two fluidically independent flow cell format) and permit imaging across the full placental cross-section.
Protocol
CREATED BY
Valentina Stanley
G4X sample preparation workflow
Samples were processed according to the following guide using a custom 306-plex placenta-focused transcript panel and a standard 16-plex immuno-oncology antibody panel:
G4X-Sample-Preparation-Guide_v5.pdf134.6MB The following protocol modifications were incorporated:
Protocol Modifications.pdf125.3KB Twenty serial sections were processed per sample to enable subsequent reconstruction of multiomic 2.5D volumes.
Data pre-processing
Initial data processing was performed using software version 3.2.4 (Mydas 25.11.0), with cell segmentation based on nuclear signal using the Cellpose nuclei model.
Acknowledgements
The core G4X sample preparation workflow was developed by a broader team at Singular Genomics, whereas the modified protocol described here was developed by the listed Singular Genomics authors in consultation with the University of California San Diego authors, who designed the transcript panel, collected and preserved tissue samples, performed clinical and histopathologic assessment for G4X tissue suitability, and carried out data analysis and interpretation.