Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues
January 10th, 2019
19.8K views
This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.
Overview
This study outlines a protocol for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations, which is crucial for understanding gene expression at the single-cell level. The techniques discussed enable detailed analysis of transcriptomes from various mammalian tissues, particularly focusing on skin and nerve tissues, and provide insights into the functional dynamics of cellular populations.
Key Study Components
Research Area
- Microbiology
- Transcriptomic analysis
- Cellular dynamics in complex tissues
Background
- Importance of individual cell analysis in understanding biological processes.
- Potential applications in studying gene expression changes due to injury or disease.
- Advances in technology enabling single-cell RNA sequencing.
Methods Used
- Dissociation of mammalian tissues into single-cell suspensions.
- High throughput single cell RNA sequencing methods.
- Flow cytometry for isolating viable cells.
Main Results
- Successful protocol for isolating high-viability single cells from skin and nerve tissues.
- Generation of high-quality cDNA libraries for sequencing.
- Comprehensive methodology reducing cell loss while preserving RNA quality.
Conclusions
- The study demonstrates an effective approach for single-cell RNA-Seq preparation.
- Findings are relevant to advancing research in cell biology and functional genomics.
Frequently Asked Questions
What tissues can be used with this protocol?
The protocol is suitable for any mammalian tissue, with specific examples provided for skin and nerve.
What is the main advantage of using single-cell RNA sequencing?
It allows for a detailed understanding of gene expression at the individual cell level within complex tissues.
How does the protocol ensure cell viability?
Quality control steps, including viability dye addition and optimized dissociation techniques, are implemented to maintain high cell viability.
How is the data from the sequencing analyzed?
Data is aligned and analyzed following a standardized bioinformatics pipeline, with results submitted to public repositories.
Who demonstrates the procedure in the video?
Elodie Labit and Wisoo Shin, trainees from the laboratory, demonstrate the procedure.
What temperature is critical during tissue processing?
It is essential to maintain the tissues at ice-cold temperatures during processing to ensure cell viability.
Why is it important to filter tissue samples?
Filtering helps to remove clumps and debris, ensuring the preparation of a single-cell suspension for accurate analysis.
Executive Industry Relevance
Single-cell transcriptomics enables biopharma R&D to resolve cellular heterogeneity in complex tissues, supporting target de-risking and mechanistic insight in disease models. This protocol provides a standardized workflow for generating high-viability single-cell suspensions from adult mammalian tissues, improving reproducibility in discovery biology. The approach enhances predictive confidence by linking molecular phenotypes to functional cell states in skin and nervous system tissues.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by resolving cell-type-specific gene expression changes in diseased or injured tissues.
- Operational Value: Supports functional target validation through unbiased profiling of cellular states and pathway activity.
- Predictive Value: Improves portfolio triage by identifying dysregulated cell populations and molecular signatures early in discovery.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream compound screening by ensuring representative and viable single-cell inputs.
- Operational Value: Standardizes tissue dissociation and filtration steps to reduce variability and improve assay reproducibility.
- Scalability: Enables platform reuse across multiple tissue types, supporting consistent readouts in phenotypic screening campaigns.
Translational & Preclinical Research
- Translational Continuity: Connects discovery-phase molecular profiling to preclinical validation through consistent cell preparation and sequencing outputs.
- Biomarker Alignment: Facilitates identification of transcriptional signatures that can serve as translational biomarkers in disease models.
- Risk-Adjusted Decisions: Supports go/no-go criteria by providing quantitative, single-cell resolution data on target engagement and cellular responses.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target identification through lead optimization, particularly when mechanistic de-risking and phenotypic screening are priorities.
- Discovery Biology: Enables hypothesis testing and pathway clarification by linking gene expression to specific cell populations in tissue contexts.
- Screening: Produces assay-ready single-cell suspensions with high viability and minimal doublets, supporting reliable compound evaluation.
- Analytics: Generates quantitative gene-barcode matrices that allow comparison of conditions and identification of differentially expressed genes.
- Translational Research: Supports preclinical continuity by providing molecular profiles that can be validated with orthogonal methods like qPCR or immunohistochemistry.
- Enterprise Reuse: Establishes a reusable capability for tissue dissociation and single-cell preparation applicable across multiple projects and disease areas.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing mechanistic ambiguity through cell-resolved transcriptomic profiling.
- Operational Value: Enhances standardization and reproducibility via defined dissociation, filtration, and viability optimization steps.
- Strategic Value: Improves capital efficiency by enabling early de-risking of targets through mechanistic insights from primary tissues.
- Portfolio Impact: Informs risk-adjusted advancement decisions by linking molecular changes to functional cell states in disease-relevant systems.
Implementation Considerations
- Requires expertise in tissue dissection, enzymatic dissociation, and flow cytometry for viable cell isolation.
- Depends on access to microfluidic platforms for droplet-based sequencing and standardized reagents for library preparation.
- Necessitates cross-team standardization of dissociation timing, enzyme concentrations, and filtration protocols to ensure consistency.
- Involves adaptation considerations when extending the protocol to tissues with varying extracellular matrix density or fragility.
- Includes practical limitations such as potential transcriptional stress from over-dissociation and the need to balance yield with RNA integrity.
Biopharma FAQs
Why does viability assessment matter for single-cell transcriptomics?
Viability assessment using dyes helps optimize dissociation conditions to preserve RNA quality and minimize cell loss, ensuring accurate representation of tissue composition. Maintaining at least 80 percent viable cells supports reliable downstream sequencing and reduces technical noise in gene expression data. This step is critical for generating high-quality cDNA libraries from primary tissues.
How does tissue dissociation impact single-cell RNA sequencing outcomes?
General dissociation is essential for achieving high cell yields and an accurate representation of tissue composition, while excessive mechanical or enzymatic stress can bias cellular populations. The protocol balances dissociation efficiency with preservation of RNA quality by optimizing incubation times and trituration steps. This approach minimizes artifacts that could confound interpretation of gene expression changes in disease or injury models.
What role does filtration play in preparing single-cell suspensions?
Filtering tissue samples through a 40 micrometer filter removes clumps and debris, ensuring a true single-cell suspension for accurate sequencing analysis. Rinsing the filter with BSA/HBSS helps recover cells and reduce loss during processing. This step is vital for preventing clogging in microfluidic systems and improving data quality in droplet-based platforms.
Why is FACS sorting used after dissociation in this workflow?
FACS isolation of viable and healthy cells using narrow-bottom tubes and BSA/HBSS washes helps remove debris and enrich for intact cells prior to sequencing. The method includes steps to prevent static and surface tension by inverting collection tubes and washing sorted cells to improve recovery. This enrichment step enhances the proportion of high-quality cells entering the single-cell controller, improving library complexity.
How does bioinformatic analysis support biological interpretation in this protocol?
Post-sequencing analysis using Seurat enables quality control through visualization of gene counts, UMIs, and mitochondrial gene percentages to identify doublets and outliers. Clustering resolution is adjusted to distinguish major cell types from transitional states, with low resolution used for heat maps and high resolution for sub-type detection. Tools like Monocle further support trajectory inference and validation of mRNA distribution norms.
Chapters in this video
0:04
Title
0:56
Dissociating Tissue (Day 1)
3:46
Isolating Viable and Healthy Cells (Day 1)
4:46
GEM (Gel Bead in Emulsion) Generation and Barcoding (Day 1)
6:17
NCBI’s GEO and SRA Submissions
7:34
Results: Bioinformatics Analysis of Single Cell Transcriptomics
9:35
Conclusion
