Sep 23, 2025
Visium sample preparation from human Middle Temporal Gyrus
- Nathan Haywood1,2,
- Beatrice Weykopf3,2,
- Zhixiang Liao3,2,
- Idil Tuncali3,2,
- Xian Adiconis1,2,
- Joshua Levin1,2,
- Clemens R. Scherzer3,2,4,5,6
- 1Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
- 3Stephen & Denise Adams Center for Parkinson’s Disease Research of Yale School of Medicine, New Haven, CT 06510;
- 4APDA Center for Parkinson Precision Medicine, Yale, New Haven, CT 06510;
- 5Department of Neurology, Yale, New Haven, CT 06510;
- 6Department of Genetics, Yale, New Haven, CT 06510
Protocol Citation: Nathan Haywood, Beatrice Weykopf, Zhixiang Liao, Idil Tuncali, Xian Adiconis, Joshua Levin, Clemens R. Scherzer 2025. Visium sample preparation from human Middle Temporal Gyrus . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5k3k6v1b/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: September 22, 2025
Last Modified: September 23, 2025
Protocol Integer ID: 227905
Keywords: ASAPCRN, Visium, Spatial transcriptomics, MTG, run visium spatial transcriptomics sample, human middle temporal gyrus
Funders Acknowledgements:
Aligning Science Acros Parkinson's
Grant ID: ASAP-000301
Abstract
This protocol describes a workflow to prepare and Run Visium Spatial Transcriptomics samples from human Middle Temporal Gyrus.
Materials
Additional materials not listed were purchased following the protocols suggestions.
Troubleshooting
Brain sample sectioning and placement
Single 10µm brain sections were prepared following strictly the Visium Spatial Protocol - Tissue Preparation guide by 10x genomics (CG000240 | Rev C, 10x Genomics) and placed onto Visium spatial gene expression slides ( #PN-1000184, 10x Genomics).
Visium spatial slides were shipped overnight collaborator on dry ice and stored at -80 °C for no more than a week before processing was continued.
Methanol Fixation H&H staining | QC Imaging
Visium slides were fixed with Methanol and subjected to H&E Staining following 10x Genomics Methanol Fixation, H&E Staining & Imaging for Visium Spatial Protocols (CG000160 | Rev B, 10x Genomics) before imaging.
Slides were imaged on an Axio Imager.Z2 microscope (Zeiss) with Metafer 5 (v3.14.150, RRID:SCR_016306 MetaSystems) automated software at 10X magnification, 1.0 CCD camera gain, and fixed lamp RGB levels.
Of note, 8 Samples were held at 4 °C > 24hours between H&E Staining and imaging du to microscope issues. No no significant difference in QC metrics were identified for these samples
Visium spatial gene expression library preparation & Sequencing parameters
Samples were processed as directed following strictly Visium spatial gene expression Reagent kits user guide (CG000239 |Rev D, 10x Genomics) using the Visium spatial Gene Expression Reagent Kit (#PN-1000186, 10xGenomics) and the Library Construction Kit (#PN-1000190, 10xGenomics). The permeabilization time was optimized as directed (CG000238 | Rev D, 10x Genomics).
Tissue optimization experiments were imaged using identical Metafer 5 settings following the H&E staining as described in step 4.
Fluorescence imaging was performed with an Agilent Technologies DNA MicroArray Scanner and analyzed using Agilent Feature Extraction (v10.5.1.1, RRID_014963, Agilent Technologies) software.
We determined the number of spots covered by each tissue section with the Loupe Browser manual alignment tool (V 5.0, RRID:SCR_018555, 10x Genomics).
Libraries were pooledfor each section, and sequenced to a depth of at least ~50,000 reads per spot on an Illumina NovaSeq with an SP flow cell with read lengths of 28 (Read 1), 90 (Read 2), 10 (Index 5), and 10 bases (Index 7).
Protocol references