Name: Comprehensive Spatial Profiling of Species-agnostic Transcriptomes via Stereo-seq (Video) | JoVE Journal
Uploaded: 2025-10-31T13:00:00.000+00:00
Duration: 10 min 22 s
Description: Learn Stereo-seq for comprehensive spatial transcriptomic profiling of host, viral, fungal, and microbiome RNA from FFPE tissue sections. This method enables high-resolution mapping while preserving tissue architecture for biomarker discovery.

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5 minutes

This research was funded in part by the Ovarian Cancer Research Alliance (OCRA 811621 and 891490), the Sie Foundation, and the Stephanie C. Stelter Endowment Fund. This research was performed in collaboration with the Flow Cytometry and Cellular Imaging Core Facility, Department of Veterinary Medicine &#38; Surgery and Advanced Genomics Technology Core, which is supported in part by the National Institutes of Health through M. D. Anderson&#39;s Cancer Center Support Grant P30 CA016672 and Jared Burks&#39; NCI&#39;s Research Specialist 1 R50 CA243707-01A1.
We also would like to thank Compete Genomics, specifically Brandon Vanderbush and Tanzeen Yusuff for technical training and troubleshooting, as well as Jia &#34;Jackie&#34; Zhao, Yongfu Wong and Erin Petrilli. The author(s) received a set of FFPE OMNI and sequencing reagents, as well as access to the T7 Early Access Program, from Complete Genomics at a discounted price. In addition, some reagents were provided free of charge for use in this study.

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</ol>

The authors declare that this work was not funded by Complete Genomics. However, Complete Genomics is supporting the costs of publication of this article.&#160; They were not given an advanced copy nor input on the content.

The Stereo-seq protocol is highly detailed and involves several critical steps that require precision, timing, and a clean environment to ensure success especially with microbiome or non-host profiling that may require specific precautions.
The use of nuclease free water during all steps is required, and the capture chip must not be touched with anything other than the sample and reagents. Exercise extreme caution when using the cassette assembly, taking time to utilize practice chips. A scrat...

Spatial-omics technologies have revolutionized the study of tissue cellular heterogeneity by enabling the simultaneous quantification of multiple targets, DNA (genomics), RNA (transcriptomics), metabolites (metabolomics), or protein (proteomics)1. The resolution of these different assays often varies, providing different limitations when measuring analytes, either with increased sparsity when measuring single cell2,3 or native spatial context4. In contrast, bulk methods such as RNA-sequencing only provide average gene expression across entire samples and obscure cellular heterogeneity2, or single-cell RNA-sequencing (scRNA-seq)2,3, which requires tissue dissociation for single-cell sequencing, often requires filtering. Therefore, these methods exclude large cells and aggregates, while bioinformatic methods try to remove fragments and doublets, all of which induce biases5,6,7. Spatial transcriptomics retains tissue architecture providing critical insights into cellular organization and interactions. Most of the non-targeted spatial sequencing methods do not have single-cell resolution, thus requiring deconvolution of spatial capture areas that containing 10 to 100 cells8,9,10. However, in the last decade, spatial transcriptomics has outpaced the needs for these methods, as there have been advancements in resolution,&#160;larger areas of capture, and the integration of multi-omics approaches, either using experimental strategy or novel techniques1,11,12.
Spatial Enhanced Resolution Omics-sequencing (Stereo-seq) provides nanoscale resolution (500 nm) and a centimeter-scale capture area, with species-agnostic whole transcriptomic capture allowing for the capture of coding, non-coding, and non-host RNA13. This method is currently the highest resolution for spatial transcriptomics and has options to provide the largest fields of view. Yet, this method has some notable limitations, including time and labor requirements of around 15 h hands-on time, spanning 4 days to complete the sequencing-ready libraries. In comparison, other sequencing-based spatial transcriptomics technologies, such as probe-based spatial transcriptomics, polyA-capture array-based spatial transcriptomics, Slide-seq, and deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) typically require 5 to 8 h of hands-on time over 2 to 3 days (Table 1). For many researchers, these advances outweigh the technical challenges and are ideal for constructing full transcriptomics spatial atlases to study disease progression at cellular and subcellular levels, particularly if layered with other technologies1.
The Stereo-seq protocol has possibilities beyond traditional mRNA transcriptomics, which relies on poly-T probes for capturing polyadenylated mRNA transcripts. These methods can be modified by adding polyadenylation to fresh tissues14 or species-specific probes for target predefined gene sets15 for spatial expression analysis. Instead, Stereo-seq uses random nucleotide probes to select diverse RNAs regardless of species, including microbial RNAs, fungal RNAs, viral RNAs, and non-polyadenylated RNAs such as enhancer RNAs, circular RNAs, and long non-coding RNAs (lncRNAs)16. Theoretically, it is possible to capture smaller RNAs or reparative elements such as small interfering RNAs (siRNAs), microRNAs (miRNAs), and transposons17. However, currently the bioinformatics and mapping of these RNA remain challenging and are often filtered out during the initial alignment&#160;, which&#160;typically&#160;uses 30 bp cutoff.
Outlined here is a detailed protocol, with our modifications for a manufacturer&#39;s random nucleotide capture on DNA nanoballs (DNB) chips to ensure better understanding and handling of a Stereo-seq protocol illustrated in Figure 1. This protocol includes strategies for molecular biologists for testing new FFPE tissues on Stereo-seq with random nucleotide capture to produce more consistent, higher yields from lower quality archived samples and fatty tissues. The outlined protocol is optimized for FFPE samples from most organs, regardless of species of origin. However, this protocol does not include current modifications needed to handle difficult tissues such as bone marrow, which require decalcification.

<table><tbody><tr><td>1.5 mL centrifuge tubes regular DNAse/RNAse</td><td>Thermo Fisher Scientific / Invitrogen</td><td>3456</td><td>To mix reagents in (other bands are fine)&amp;nbsp;</td></tr><tr><td>100% Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td>Molecular grade</td></tr><tr><td>15mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Thermo Fisher Scientific / Invitrogen</td><td>339650</td><td>Used for aliquots&amp;nbsp;</td></tr><tr><td>20x Concentrate&amp;nbsp;Saline Sodium Citrate (SSC) Buffer</td><td>Sigma-Aldrich</td><td>S6639-1L</td><td>Used to make 5X SSC for staining and 1X SSC for wash steps and coverslip removal.</td></tr><tr><td>2100 Bioanalyzer instrument</td><td>Agilent</td><td>Discountinued</td><td>Instument: Bioanalyzer</td></tr><tr><td>3 Well Chamber, removable</td><td>Ibidi</td><td>80381</td><td>Silicone chamber to reduce volumes during rehydration and methanol fixation.</td></tr><tr><td>405nm UV Light Source</td><td>Various</td><td>N/A</td><td>For curing UV resin</td></tr><tr><td>50mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Thermo Fisher Scientific / Invitrogen</td><td>339652</td><td>Used for aliquots and for deparaffinization&amp;nbsp;</td></tr><tr><td>70% Sterile Isopropanol Alcohol</td><td>Texwipe&amp;nbsp;</td><td>TX3270</td><td>For surface decontamination&amp;nbsp;</td></tr><tr><td>Agilent High Sensitivity DNA Kit</td><td>Agilent</td><td>5067-4626</td><td>For Bioanalyzer characterization of cDNA and libraries</td></tr><tr><td>Agilent RNA 6000 Pico Kit</td><td>Agilent</td><td>5067-1513</td><td>For Bioanalyzer characterization of RNA</td></tr><tr><td>Aluminum Foil</td><td>Various</td><td>N/A</td><td>To cover slide during ssDNA staining.</td></tr><tr><td>Beckman Coulter SPRIselect</td><td>Beckman Coulter Life Sciences&amp;nbsp;</td><td>B23317/B23318/B23319</td><td>SPRI Beads cDNA and Library Clean up</td></tr><tr><td>Bleach</td><td>Various</td><td>N/A</td><td>Cleaning workstation removing microbiome contamination</td></tr><tr><td>CONSTIX Sealed Foam Swabs</td><td>Contec</td><td>19161023</td><td>For precise resin cleaning</td></tr><tr><td>Coverslip</td><td>Sigma-Aldrich</td><td>BR470045-2000EA</td><td></td></tr><tr><td>Desiccant Packs</td><td>Various</td><td>N/A</td><td>RNase free, dust free desiccant packs.</td></tr><tr><td>Disposable Face Mask</td><td>Thermo Fisher Scientific / Invitrogen</td><td>12-888-001</td><td>Protection, RNAse contamination and &amp;amp; sterility&amp;nbsp;</td></tr><tr><td>DNA Erase Wipes</td><td>Sigma-Aldrich</td><td>L9060-250EA</td><td>Decontamination by wiping off surfaces (wet wipes)</td></tr><tr><td>DNA LoBind Tubes 1.5mL</td><td>Eppendorf&amp;nbsp;</td><td>22431021</td><td>Used for collection of cDNA and library post amplifications.</td></tr><tr><td>DNA LoBind Tubes 2mL</td><td>Eppendorf&amp;nbsp;</td><td>22431048</td><td>Used for collection of cDNA before amplification.</td></tr><tr><td>DNBSEQ OneStep DNB Make Reagent Kit</td><td>Complete Genomics</td><td>940-001891-00</td><td>Reagents for DNA nanoball (DNB) preparation and amplification</td></tr><tr><td>DNBSEQ-T7RS Cleaning Reagent Kit</td><td>Complete Genomics</td><td>940-001903-00</td><td>Cartridge for washing after sequencing complete</td></tr><tr><td>DNBSEQ-T7RS DNB Load Reagent Kit</td><td>Complete Genomics</td><td>940-001894-00</td><td>Reagents and plate to load DNBs to flow cell</td></tr><tr><td>DNBSEQ-T7RS Sequencing Flow Cell</td><td>Complete Genomics</td><td>940-001902-00</td><td>1 lane/flow cell</td></tr><tr><td>DNBSEQ-T7RS Stereo-seq Visualization Reagent Kit</td><td>Complete Genomics</td><td>940-001893-00</td><td>The cartridge and reagents for sequencing.</td></tr><tr><td>DNBSEQ-T7RS system</td><td>Complete Genomics</td><td></td><td>This stereo-seq visualization set utilizes DNBSEQ technology. A sequencing run starts with the hybridization of a DNA anchor, then a fluorescent probe is attached to the DNA Nanoball (DNB) using combinatorial probe anchor sequencing (cPAS) chemistry. Finally, the high-resolution imaging system captures the fluorescent signal. After digital processing of the optical signal, the sequencer generates high-quality and accurate sequencing information.</td></tr><tr><td>Dust-Off Compressed Air</td><td>Matin</td><td>M-6318</td><td>Important to use this brand or one with similar propellant&amp;nbsp;</td></tr><tr><td>Edge-Rite Microtome Blades</td><td>Thermo Fisher Scientific / Invitrogen</td><td>4280L</td><td>Histology - Used when sectioning samples</td></tr><tr><td>Explosion proof Freezer</td><td>Various</td><td>N/A</td><td>To chill methanol before and during methanol fixation step.</td></tr><tr><td>Histology Brushes</td><td>Various</td><td>N/A</td><td>Histology - Used when sectioning samples</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>2104-50ML</td><td>For diluting premuliablization enzyme in 0.01 N HCl (pH 2). Standard stock concentration for HCl solution is 0.1 N. </td></tr><tr><td>Imaging System</td><td>various</td><td>N/A</td><td>GC Stomics Imager, Lecia, Evo Revolution</td></tr><tr><td>Invitrogen RNaseZap RNase Decontamination Solution</td><td>Thermo Fisher Scientific / Invitrogen</td><td>AM9780</td><td>Cleaning workstation removing RNAse contamination</td></tr><tr><td>Kimtech Delicate Task Wipers&amp;nbsp;</td><td>Kimtech</td><td>34155</td><td>To dry benches, equipment, pipettes, etc.</td></tr><tr><td>Labcoat</td><td>Various</td><td>N/A</td><td>Personal Protection Equipment (PPE)</td></tr><tr><td>Lens Paper</td><td>Thermo Fisher Scientific / Invitrogen</td><td>11-997</td><td>Various works for drying slides without sheading</td></tr><tr><td>Lookout DNA Erase Wipes</td><td>Sigma-Aldrich</td><td>L9060</td><td>Cleaning workstation removing PCR contamination</td></tr><tr><td>Magentic Separation Rack 5uL - 0.2&amp;nbsp;</td><td>Permagen Labware</td><td>MSRLV08</td><td>For bead sepration in PCR tubes</td></tr><tr><td>Magjet Rack 12x1.5 mL</td><td>Thermo Fisher Scientific / Invitrogen</td><td>MR02</td><td>For bead sepration in 1.5 mL tubes</td></tr><tr><td>Manual Rotary Microtome</td><td>Leica</td><td>RM2235</td><td>Histology - Used when sectioning samples</td></tr><tr><td>Methonal &amp;ge;99.9%, suitable for immunofluorescence, HPLC</td><td>Sigma-Aldrich</td><td>34860</td><td>For fixation (Needs to be HPLC Grade)</td></tr><tr><td>Micro-dissecting Forceps</td><td>Sigma-Aldrich</td><td>F4142-1EA</td><td>Histology - Used when sectioning samples</td></tr><tr><td>Micropipette</td><td>Various</td><td>N/A</td><td></td></tr><tr><td>Molecular Grade Nuclease Free Water Non-DPEC Treated Water</td><td>Various</td><td>N/A</td><td>Dilutions and elution of beads</td></tr><tr><td>Nitrile Gloves</td><td>Various</td><td>N/A</td><td>PPE</td></tr><tr><td>Oven</td><td>Various</td><td>N/A</td><td>To bake chip</td></tr><tr><td>PCR 0.2 mL Tubes with attached caps</td><td>Various</td><td>N/A</td><td>For PCR applications</td></tr><tr><td>PCR covers&amp;nbsp;</td><td>Various</td><td>N/A</td><td>Replacement if adhesive on provided cover is weak</td></tr><tr><td>PCR Hood &amp;amp; Workstation</td><td>Mystaire</td><td>MY-PCR24-010</td><td>For pre-PCR (RNA to cDNA) steps</td></tr><tr><td>pH Meter</td><td>Various</td><td>N/A</td><td>pH Meter to confirm HCl pH.</td></tr><tr><td>Poly-L-Lysine solution</td><td>Sigma-Aldrich</td><td>A005-C</td><td>Optional to increase tissue adhesion to chip</td></tr><tr><td>Qubit 4 Fluorometer</td><td>Thermo Fisher Scientific / Invitrogen</td><td>Q33226</td><td>Insuterment; DNA quantiy measurement</td></tr><tr><td>Qubit dsDNA Quantification Assay Kits</td><td>Thermo Fisher Scientific / Invitrogen</td><td>Q32851</td><td>For measuring cDNA and library quantifications</td></tr><tr><td>Qubit ssDNA Assay Kit</td><td>Thermo Fisher Scientific / Invitrogen</td><td>Q10212</td><td>ssDNA staining (Only dye used)</td></tr><tr><td>RNAse-free Ice Block</td><td>Various</td><td>N/A</td><td>Histology - Used when sectioning samples to hydrate blocks before sectioning</td></tr><tr><td>RNaseZap RNase Decontamination Solution</td><td>Thermo Fisher Scientific / Invitrogen</td><td>AM9782</td><td>A surface decontamination solution that destroys RNAses</td></tr><tr><td>RNeasy FFPE Kit</td><td>Qiagen</td><td>73504 &amp;amp; 19093</td><td>For RNA Extraction &amp;amp; DV200 measurement&amp;nbsp;</td></tr><tr><td>Sculpt Ultra White Resin</td><td>Siraya Tech</td><td>N/A</td><td>High-temperature resistant UV curable resin</td></tr><tr><td>Stereo-seq 16 Barcode Library Preparation Kit</td><td>STOmics / Complete Genomics</td><td>111KL160</td><td>For constructing Stereo-seq OMNI FFPE Library</td></tr><tr><td>Stereo-seq FFPE Accessory Kit</td><td>STOmics / Complete Genomics</td><td>310AK002</td><td>Spatial OMNI accessory parts of 211SN114 kit</td></tr><tr><td>Stereo-seq N-chip Slide (1cm x 1cm)</td><td>STOmics / Complete Genomics</td><td>210CN114</td><td>Spatial OMNI chip part of 211SN114 kit</td></tr><tr><td>Stereo-seq PCR Adaptor&amp;nbsp;</td><td>STOmics / Complete Genomics</td><td>301AUX001</td><td>For PCR hybridization steps modified to fit three similar to PCRmax Situ Hybridization Adapter</td></tr><tr><td>Stereo-seq Transcriptomics N Kit</td><td>STOmics / Complete Genomics</td><td>211KN114</td><td>Spatial OMNI regent part of 211SN114 kit</td></tr><tr><td>Surgical Design General Purpose Industrial Razor Blade</td><td>Thermo Fisher Scientific / Invitrogen</td><td>13-812-236</td><td>Histology - Used when sectioning samples</td></tr><tr><td>TE Buffer Solution pH 8.0</td><td>Sigma-Aldrich</td><td>8890</td><td>For eluting cDNA and libraries</td></tr><tr><td>Thermal Cyclers</td><td>BioRad / various</td><td>1861096 /12015392 / various</td><td>PCR Machine with manual lid or deep well options (T100 Thermal Cycler / PTC Tempo Deepwell Thermal Cycler etc)</td></tr><tr><td>Thermo Scientific Orion All-in-One pH Buffer Kits</td><td>Thermo Fisher Scientific / Invitrogen</td><td>13-624-500</td><td></td></tr><tr><td>Tissue Flotation Bath - Digital XH-1003</td><td>Premiere</td><td>NC0779538</td><td>Histology - Used when sectioning samples</td></tr><tr><td>UV-protective Eyewear</td><td>Various</td><td>N/A</td><td>For safety during UV curing</td></tr><tr><td>Xylenes</td><td>Sigma-Aldrich</td><td>247642</td><td>For deparaffinization</td></tr></tbody></table>

comprehensive spatial profiling of species-agnostic transcriptomes via stereo-seq

Comprehensive Spatial Profiling of Species-agnostic Transcriptomes via Stereo-seq

The spatial composition of cells within the host, as well as the bacteria or viral loads within the tissue, can impact the interaction of cell types and analytes that drive the cell through cell-type-specific processes. Stereo-seq for FFPE tissues uses random priming to spatial barcodes, which is different from standard spatial transcriptomics methods, which use A&#39;tailing to capture messenger RNA (mRNA) or probe-based to capture species-specific transcripts. These methods do not embrace the more current knowledge about the impact and importance of other types of RNA from long non-coding RNA, mitochondrial RNA, microRNA, or&#160;other species,&#160;RNA, such as microbial, viral, and fungal. Outlined here is a step-by-step procedure from tissue sectioning through library preparation for spatial species-agnostic stereo-seq application with RNA detection using a randomer probe methodology, which is compatible with formalin fixed paraffin embedded (FFPE) tissues. This Stereo-seq method has a random capture bead of 0.22 &#181;m in size that reoccurs in an array every 0.5 &#181;m, allowing for subcellular resolution from a sequencing-based technology. As this method detects both host and non-host RNA, the protocol requires specific considerations to allow for the determination of what is inside the tissue versus what was deposited on the tissue during the collection, preservation, cutting, and detection process (i.e., environmental and handling contaminants). Lastly, this protocol allows one&#160;to have high (single-cell) level resolution of multi-RNA species within a spatial context, providing insight into the intra- and inter-actome of cell types and pathological species.

The data below demonstrates the ability to perform single-cell segmentation using the SAW pipeline and mapping to non-poly adenylated RNAs such as tRNA (TRDMT1) from an FFPE section. The unbiased data from Stereo-seq allow one to examine the importance of not just mRNAs but also tRNAs, rRNAs, and non-host RNAs (if pertinent). Using the direct output from the SAW pipeline16 and the stereopy for quality assessment of the Stereo-seq output in Figu...

This article presents a protocol to perform comprehensive spatial transcriptomic profiling of host, viral, fungal, and microbiome RNA from formalin-fixed paraffin-embedded tissue sections using Stereo-seq, which enables high-resolution mapping of diverse transcriptomes while preserving tissue architecture.

The spatial composition of cells within the host, as well as the bacteria or viral loads within the tissue, can impact the interaction of cell types and ...

Our research focuses on characterizing the ovarian tumor microenvironment in detail to identify predictive and therapeutic biomarkers. For example, we would like to find markers to predict chemo response and chemo resistance or novel therapeutical targets for second line-treatments. The experimental challenges include the sample handling, the quality assessment of the sample, and the length of the time for processing a sample.While on the data analysis side, the challenges include things like the sparsity of the data, similar to other single cell processes or and also the mapping of areas, especially repetitive regions. After removing the stereo-sequencing N-Chip from its packaging, record the chip identification number. Without touching the N-Chip surface, clean the glass slide with 100%ethanol.Using a micro pipette, carefully apply 10 microliters of ultraviolet curable ceramic resin around the edge of the stereo-sequencing N-Chip. Use a sealed foam swab to remove excess resin, leaving only a thin line around the chip edge. Place the slide into the ultraviolet curing device, ensuring the chip surface is not touched.Now place the resin-applied chip on top of an opaque mask on the 405-nanometer ultraviolet light source to illuminate the back of the slide, and cure for five minutes. After curing, use sealed foam swabs soaked in 100%molecular-grade ethanol, then 70%ethanol, and finally, nuclease-free water to clean around the resin-applied edge. Submerge the chip three times in fresh nuclease-free water using a 50-milliliter conical tube.Using compressed air, dry the chip with the full force of air at an angle of approximately 60 to 80 degrees, moving diagonally across the surface from about five centimeters away. Next, apply 150 microliters of Poly-L-Lysine solution evenly across the entire chip surface, and incubate the chip for 10 minutes at room temperature. Then remove the Poly-L-Lysine solution from the chip.After the second wash, carefully dry the edges of the slide without touching the chip surface. Firmly dry the chip using compressed air as demonstrated earlier. While sectioning the FFPE tissue, do not touch the chip directly, and only cover 80%of the chip with tissue.To prepare two microliters of single-stranded DNA staining solution in SSC buffer, dilute the Qubit single-stranded DNA reagent with 5X SSC buffer. Create a master mix of staining solution using a one to 20 dilution of ribonuclease inhibitor, a one to 200 dilution of the single-stranded DNA solution, and the remaining volume with 5X SSC buffer. Add 150 microliters of staining solution to each chip and incubate in the dark at room temperature for five minutes.Then gently remove the staining solution from the corner of each chip using a pipette. After washing the chip twice with 0.1X SSC buffer for 10 to 15 seconds, carefully dry the edges of the slide. Add approximately three to five microliters of glycerol onto the slide to mount it.Drop a coverslip from about one centimeter above the slide, keeping it level, and ensuring it does not touch the chip surface. Capture fluorescent images of single-stranded DNA-stained tissue using a wide-field microscope, ensuring 10%overlap between image tiles. Stitch the acquired images in image processing software such as ImageJ by first utilizing theoretical overlap to generate approximate tile positions, then applying pixel matching computational overlap to fine-tune the tiling.Process the stitched images in StereoMap software, and confirm that image quality control passes before continuing. Submerge the stereo-sequencing slide in 30 milliliters of 0.1X SSC buffer until the coverslip detaches. Ensure the FFPE decrosslinking reagent is at room temperature and inspect it for particles.Turn on the thermocycler and set it to 30 degrees Celsius for equilibration, 95 degrees Celsius for a 30-minute incubation, and infinite hold at four degrees Celsius. Assemble the cassette gasket and place the stereo-sequencing chip slide into the cassette. Then add FFPE decrosslinking reagent into the well of the stereo-sequencing slide cassette, apply sealing tape to the cassette and confirm it is tightly sealed.Start the 30-minute incubation at 95 degrees Celsius. After 10 minutes, place 25 milliliters of methanol in a 50-milliliter container in a 20 degrees Celsius freezer, preparing about one milliliter per slide for later use. After removing the chip from the thermocycler, transfer the slide cassette to the bench and peel off the sealing tape.Using a pipette, remove and discard the FFPE decrosslinking reagent from the cassette. Once the reagent has been completely removed, detach the cassette and gasket and discard them. Under a sterile fume hood, dry the edge of the slide, and apply a new silicone chamber if needed.Add 500 microliters of chilled methanol from the 20 degrees Celsius freezer, ensuring the entire section is submerged. Prewarm PR solution on a 37 degrees Celsius dry block for 10 minutes before use. After fixation, in a fume hood, wipe off any excess methanol from the slide and wait for the remaining methanol on the chip to evaporate.Assemble a new cassette end gasket. Once evaporation is complete, place the stereo-sequencing chip slide into the cassette. Add 200 microliters of prewarmed permeablization reagent to the chip.Apply sealing tape to the stereo-sequencing slide cassette and ensure it is tightly sealed. Perform reverse transcription and then CDNA release and purification following the steps as shown here. Preheat nuclease-free water to 37 degrees Celsius.Remove the seal from the chip. Pipette vigorously in each corner and in the center of the chip over the tissue without scraping or touching it. Collect all the complimentary DNA release mix from the chip and transfer it into a 1.5 or 2.0-milliliter centrifuge tube.Add 350 microliters of preheated nuclease-free water directly onto the chip surface. Pipette vigorously with a smaller pipette, angling the tip to apply sheer force without touching the tissue or scraping the chip. Combine the wash with the 400 microliters of complimentary DNA release mix collected earlier, ensuring that only material from a single chip is combined.Place 100 microliters of nuclease-free water on the chip. Seal the stereo-sequencing slide, and store it in the refrigerator until the end of the protocol. Single-cell segmentation using the SAW pipeline was achieved on FFPE sections, enabling mapping to non-polyadenylated RNAs such as tRNA, TRDMT1.Interactive spatial visualization showed putative cell types using the proprietary software StereoMap, with clusters displayed in various colors across a zoomed-in region of the tissue. Whole tissue expression of the non-polyadenylated RNA TRDMT1 was visualized using StereoPi. The adaptations of the protocol address issues we had in full transcriptomics spatial profiling of fatty and fragile tissues by improving tissue adhesion, RNA accessibility, and handling for stereo-seqs on these difficult tissue types.Stereo-seq currently provides higher resolution than other spatial transcriptomic methods with a resolution of 500 nanometers compared to two microns. It also provides unbiased capture needed for the study of the microbiome, viral, and non-polyadenylated transcripts.

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JoVE Journal

Cancer Research

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RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

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Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

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Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms

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Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

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Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

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Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

The protocol presented here describes a complete pipeline to analyze RNA-sequencing transcriptome data from raw reads to functional analysis, including quality control and preprocessing steps to advanced statistical analytical approaches.

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 µm.

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

This study presents a spatial Trekker–CUT&amp;Tag multiome protocol combining direct spatial barcoding of a tissue section with single-cell multiome analysis. This approach enables spatially resolved single-cell profiling of gene expression and the histone H3K27ac modification simultaneously, offering mechanistic insights into gene regulation in the context of tissue microanatomy.

Spatially Resolved, Integrated Single-Cell Multiomic Profiling of the Transcriptome and Epigenomic Targets in Frozen Tissue Sections

Learn Stereo-seq for comprehensive spatial transcriptomic profiling of host, viral, fungal, and microbiome RNA from FFPE tissue sections. This method enables high-resolution mapping while preserving tissue architecture for biomarker discovery.

Comprehensive Spatial Profiling of Species-agnostic Transcriptomes via Stereo-seq

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