Dec 20, 2024

Public workspaceSpatial RNA sequencing of human ganglia

DOI
dx.doi.org/10.17504/protocols.io.j8nlk8jb6l5r/v1
  • 1Department of Neuroscience, Center for Advanced Pain Studies, The University of Texas at Dallas
  • PRECISION Human Pain Network
Ayesha Ahmad
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk8jb6l5r/v1
Protocol Citation: Stephanie Shiers, Diana Tavares-Ferreira, Ishwarya Sankaranarayanan, Khadijah Mazhar, Theodore Price 2024. Spatial RNA sequencing of human ganglia. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8jb6l5r/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 16, 2024
Last Modified: December 20, 2024
Protocol Integer ID: 107725
Keywords: human, Visium, DRG, spatial, sequencing
Funders Acknowledgements:
National Institutes of Health
Grant ID: U19NS130608
National Institutes of Health
Grant ID: NS111929
National Institutes of Health
Grant ID: NS065926
Abstract
This protocol covers spatial RNA sequencing of human dorsal root ganglia sections from fresh frozen samples using the 10X Visium Spatial Gene Expression kits, a poly-A tail based mRNA sequencing assay. The final raw data output are count matrices of gene expression.
Materials
Tissue preparation and sectioning
  1. Fresh frozen human ganglia
  2. Metal cryomold (Ted Pella, 27276-5)
  3. OCT (Fisher, 23-730-571)
  4. Visium spatial gene expression slide (10X Genomics, PN-2000233)

Fixation, Hematoxylin and Eosin Staining
  1. 100% methanol (Fisher Chemical, A456-212)
  2. Hematoxylin (Agilent Tech, S330930-2)
  3. Eosin (Sigma, HT110216)
  4. Tris base (Thermo Fisher Scientific, BP152-500)
  5. Nuclease-free water (Invitrogen, AM9937).
  6. 100% Acetic Acid (Millipore Sigma, A6283).
  7. 0.2 μm Corning 250mL Vacuum System (Corning, 430771)
  8. Bluing Buffer (Agilent Tech, CS70230-2)
  9. Glycerol (Sigma Aldrich, G9012)

Library construction
  1. Visium Spatial Gene Expression Slide & Reagent Kit, 16 rxns PN-1000184

Equipment:
  1. Cryostat for sectioning
  2. Bio-Rad T100 Thermal Cycler with Thermocyler Adaptor
  3. Microscope for brightfield imaging
  4. Sequencing system
Safety warnings
Safety training courses on Bloodborne Pathogens, Biological Safety, Chemical Hygiene, Compressed Gases, Biological Waste Management, Personal Protective Equipment, Autoclave and Media Kitchens, General Laboratory Safety, and Cryogen Safety are required by UTD and can be taken through its BioRAFT system.

Safety Training related to Biomedical Research with Human Subjects is required by UTD and can be taken through its CITI program.
Ethics statement
All human tissue recovery, research and data handling is performed in accordance with the University of Texas at Dallas Institutional Review Board (IRB) regulations.
Before start
Ensure proper personal protective equipment (PPE) is worn and institutionally required safety guidelines are followed.
Tissue preparation and sectioning
Tissue preparation and sectioning
Human ganglia are procured fresh frozen and stored at -80 C.
Tissue is embedded in OCT for cryosectioning. For a detailed embedding protocol, see steps 37-42 of dx.doi.org/10.17504/protocols.io.kqdg32qr1v25/v1.
Embedded tissue is sectioned onto slides provided for 10X Genomics Visium spatial gene expression assay (Visium Spatial Slides), following Demonstrated Protocol CG000240 (section 2). Sections are 10 µm thick and trimmed before being adhered to the slide to fit capture areas.
Fixation, Hematoxylin and Eosin (H&E) staining, and imaging
Fixation, Hematoxylin and Eosin (H&E) staining, and imaging
Slides are fixed as described in Demonstrated Protocol CG000160 (section 1.2).
Slides are H&E stained as described in Demonstrated Protocol CG000160 (section 1.3).
Slides are coverslipped as described in Demonstrated Protocol CG000160 (Appendix) using a glycerol-based mounting medium.
Slides are mosaically imaged at 10X using default brightfield settings on an Olympus vs120 slide scanner
After imaging, the coverslip is removed by soaking slides in 3X SSC buffer as described in Demonstrated Protocol CG000160 (Appendix). Slide is then immediately processed for Library Construction.
Library Construction and Sequencing
Library Construction and Sequencing
Library construction is performed following User Guide CG000239 for the Visium Spatial Gene Expression Reagent Kit.
Permeabilization & Reverse Transcription: Permeabilization time needs to be optimized for each tissue type used. Optimized time for human DRG tissue is 12 minutes as published in 10.1126/scitranslmed.abj8186.
Second Strand Synthesis & Denaturation: no changes or optimization to protocol
cDNA Amplification & QC: cDNA amplification steps are performed with the optimal cycle numbers as determined per run, per protocol instructions. Amplification and QC are performed by UTD Genome Center as described in protocol.
Spatial Gene Expression Library Construction: no changes or optimization to protocol. These steps are performed by UTD Genome Center
Sequencing: sequencing is performed at UTD Genome Center.
Analysis
Analysis
Raw sequencing files are processed with the 10X Genomics SpaceRanger pipeline to generate count matrices of gene expression per Visium barcode.
Visium sections are examined on Loupe Browser (10X Genomics) to select for barcodes of interest
Gene expression analysis of barcodes is done using R (version 4.3.3) for downstream processing of the SpaceRanger output.