Name: Uitgebreide ruimtelijke profilering van soortagnostische transcriptomen via stereo-seq (Video) | JoVE Journal
Uploaded: 2025-10-31T13:00:00.000+00:00
Duration: 10 min 22 s
Description: Leer ruimtelijke transcriptoomprofilering van weefsel met Stereo-seq voor identificatie van biomarkers. Deze methode behoudt weefselarchitectuur en helpt bij het vinden van voorspellende markers voor chemotherapierespons.

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</ol>

<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)&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&nbsp;</td></tr><tr><td>20x Concentrate&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&nbsp;</td></tr><tr><td>70% Sterile Isopropanol Alcohol</td><td>Texwipe&nbsp;</td><td>TX3270</td><td>For surface decontamination&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&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; sterility&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&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&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&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&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&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 &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&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; 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>Q32

uitgebreide ruimtelijke profilering van soortagnostische transcriptomen via stereo-seq

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

Uitgebreide ruimtelijke profilering van soortagnostische transcriptomen 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 ...

comprehensive-spatial-profiling-species-agnostic-transcriptomes-via

Research

JoVE Journal

Cancer Research

Kankeronderzoek

Deze video toont de basisstappen voor het uitvoeren van een volledige transcriptoomanalyse op gedissecteerde vogelembryonale ruggenmergmonsters na transfectie met in ovo elektroporatie.

RNA-Seq Analyse van differentiële genexpressie in Geëlektroporeerde Chick Embryonale Spinal Cord

RNA-sequencing en bioinformatica analyses werden significant verschillend tot expressie en transcriptiefactoren in Lin-CD34 + en CD34-Lin subpopulaties van muis EMLcells identificeren. Deze transcriptiefactoren kunnen een belangrijke rol spelen bij het bepalen van de switch tussen zelfvernieuwende Lin-CD34 + en gedeeltelijk gedifferentieerd Lin-CD34-cellen.

Identificatie van Key factoren die de Self-vernieuwing en differentiatie in EML Hematopoietic voorloper cellen door RNA-sequencing Analyse

Dit protocol schetst een vergelijkende de novo transcriptoomsamenstelling en annotatiestroom voor beginnende bioinformatici. De workflow is volledig gratis beschikbaar via CyVerse en verbonden door de Data Store. Er worden opdrachtregel- en grafische gebruikersinterfaces gebruikt, maar alle benodigde code is beschikbaar om te kopiëren en te plakken.

CyVerse Resources gebruiken voor<em&gt; De Novo</em&gt; Vergelijkende transcriptomics van onderverdiende (niet-model) organismen

Dit protocol beschrijft het combinatorische gebruik van ChIP-seq, 4sU-seq en totaal RNA-seq ribosoom profilering voor cellijnen en primaire cellen. Hierdoor wijzigingen bijhouden in transcriptiefactor bindend, DOVO transcriptie, RNA-verwerking, omzet en vertaling na verloop van tijd, en de algemene gang van zaken in geactiveerd en/of snel veranderende cellen weer te geven.

Real-time analyse van transcriptiefactor bindend, transcriptie, vertaling en omzet globale u gebeurtenissen wilt weergeven tijdens de activatie van de cellulaire

Hier presenteerden we een multiplexed single cell mRNA sequencing methode om het profiel van genexpressie in de muis embryonale weefsels. De op droplet gebaseerde enkelvoudige cel mRNA-sequencing (scRNA-SEQ) methode in combinatie met multiplexing strategieën kan afzonderlijke cellen van meerdere monsters tegelijkertijd profiel, wat de kosten van de reagens aanzienlijk verlaagt en experimentele batch-effecten minimaliseert.

Multiplexed single cell mRNA sequentieanalyse van embryonale cellen van de muis

Hier tonen de auteurs het nut van MULTI-seq voor fenotypering en daaropvolgende gepaarde scRNA-seq en scATAC-seq bij het karakteriseren van de transcriptomische en chromatinetoegankelijkheidsprofielen in retina.

Multiplexed analyse van retinale genexpressie en chromatinetoegankelijkheid met behulp van scRNA-Seq en scATAC-Seq

Het hier gepresenteerde protocol beschrijft een complete pijplijn om RNA-sequencing transcriptoomgegevens te analyseren, van ruwe reads tot functionele analyse, inclusief kwaliteitscontrole en voorbewerkingsstappen tot geavanceerde statistische analytische benaderingen.

Transcriptoomanalyse met hoge doorvoer voor het onderzoeken van gastheer-pathogeeninteracties

Hier beschrijven we een protocol voor het verfijnen van interessante regio's (ROI's) voor Spatial Omics-technologieën om de micro-omgeving van de tumor beter te karakteriseren en specifieke celpopulaties te identificeren. Voor proteomics-assays kunnen geautomatiseerde aangepaste protocollen de ROI-selectie begeleiden, terwijl transcriptomics-assays kunnen worden verfijnd met behulp van ROI's zo klein als 50 μm.

Ruimtelijke profilering van eiwit- en RNA-expressie in weefsel: een benadering om virtuele microdissectie te verfijnen

Dit protocol presenteert een reproduceerbare workflow voor het analyseren van ruimtelijke transcriptomics-gegevens en begeleidt gebruikers bij de acquisitie van openbare gegevens en Seurat-gebaseerde kwaliteitscontrole, gevolgd door integratie, detectie van ruimtelijke kenmerken, deconvolutie van celtypen, annotatie van regio's van belang en analyse van cel-celcommunicatie, inclusief praktische controlepunten die een transparante uitvoering ondersteunen.

Een praktische workflow voor de analyse van ruimtelijke transcriptomics-gegevens: van gegevensverwerving tot geavanceerde analyses

Deze studie presenteert een ruimtelijk Trekker–CUT&amp;Tag multioomprotocol dat directe ruimtelijke streepjescoding van een weefselsectie combineert met enkelcel-multioomanalyse. Deze aanpak maakt ruimtelijk opgeloste enkelcelprofilering van genexpressie en de histon H3K27ac-modificatie mogelijk gelijktijdig, wat mechanistische inzichten biedt in genregulatie in de context van weefselmicroanatomie.

Ruimtelijk opgeloste, geïntegreerde single-cell multiomic profilering van het transcriptoom en epigenomische doelen in bevroren weefselsecties

Leer ruimtelijke transcriptoomprofilering van weefsel met Stereo-seq voor identificatie van biomarkers. Deze methode behoudt weefselarchitectuur en helpt bij het vinden van voorspellende markers voor chemotherapierespons.

Uitgebreide ruimtelijke profilering van soortagnostische transcriptomen via stereo-seq

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