This webinar, presented by José Morachis of NanoCellect Biomedical and Dr. Brian Fritz of 10x Genomics, details the critical importance of upstream sample preparation for successful single-cell genomic analyses. Morachis introduces the NanoCellect Wolf cell sorter, a microfluidic-based platform that uses exceptionally low pressure (<2 psi) to gently isolate target cells, remove debris and dead cells, and improve overall sample quality. He provides evidence that this gentle sorting process increases cell viability and leads to higher quality downstream data from the 10x Genomics platform, demonstrated by increased gene and UMI counts and more resolved cell clusters in sorted samples.
Dr. Fritz then explains how high-quality input samples enable advanced "multi-omic cytometry" applications. This approach simultaneously measures multiple analytes from each single cell, including mRNA for gene expression, cell surface proteins via oligonucleotide-tagged antibodies (feature barcoding), T-cell receptor sequences, and antigen specificity using tagged peptide-MHC dextramers. He presents a case study on identifying rare antigen-specific T-cells, emphasizing that pre-sorting is "absolutely critical" for the efficiency and feasibility of such experiments, allowing for the comprehensive linkage of a T-cell's immunophenotype, clonal identity, and functional state.
Enhancing Single-Cell Genomics: The Critical Role of Sample Preparation and Multi-Omic Analysis
2:47The Challenge of Single-Cell Analysis: The webinar begins by highlighting the importance of high-quality starting material for single-cell sequencing. Poor sample preparation—with debris, dead cells, or non-target cells—can lead to clogged microfluidic systems, degraded RNA, and inaccurate, costly results.
8:20A Gentle Sorting Solution: José Morachis introduces the NanoCellect Wolf cell sorter, an easy-to-use, microfluidic-based system designed to improve sample prep. It uses sterile, disposable cartridges to prevent cross-contamination.
10:49High Viability Through Low Pressure: The Wolf sorter's key feature is its extremely low-pressure sorting mechanism (<2 psi), which is significantly gentler than traditional FACS systems (up to 70 psi). This results in higher cell viability, which is especially important for sensitive cell types like stem cells and neurons.
11:17Improving Data and Reducing Costs: By sorting for a pure population of live, target cells, researchers can increase the percentage of useful cells in their sample, improving data quality and reducing the effective cost per cell in a sequencing experiment.
16:01Isolating Rare Cell Populations: For targets that are rare (e.g., <1% of the population), a two-step workflow is presented: first, an initial bulk enrichment (using a method like Akadeum beads), followed by gentle sorting on the Wolf to achieve high purity (>90%) of the rare cell type.
17:57Handling Difficult Tissues: For samples that are hard to dissociate into single cells, such as brain tissue or plants, isolating and sorting nuclei is presented as a viable and effective alternative to get clean input for genomic analysis.
20:57Proof of Better Data: An experiment comparing sorted vs. unsorted PBMCs for a 10x Genomics run shows that the sorted sample yielded superior data, including higher median genes per cell, higher UMI counts, and the resolution of nine distinct cell clusters compared to only seven in the unsorted sample.
26:17Introduction to Multi-Omic Cytometry: Dr. Brian Fritz explains how 10x Genomics technology enables multi-omic cytometry, where multiple types of biological information (analytes) are measured from the same single cell.
35:18Simultaneous Measurement of RNA and Protein: The platform can measure both gene expression (mRNA) and cell-surface protein levels by using antibodies tagged with oligonucleotide barcodes (a technique known as "feature barcoding").
39:30Defining T-Cell Specificity: The multi-omic approach can be extended to immunology to simultaneously identify a T-cell's gene expression, its unique T-cell receptor (TCR) sequence, and its specific antigen target by using oligo-tagged peptide-MHC dextramers.
44:30Sorting is "Absolutely Critical" for Rare Cell Discovery: Dr. Fritz emphasizes that for complex experiments targeting very rare cells, such as antigen-specific T-cells, upstream cell sorting is not just helpful but "absolutely critical" to efficiently find the cells of interest and make the experiment cost-effective.
45:31Case Study: High-Resolution Immune Profiling: A detailed experiment is shown where CMV-specific T-cells were sorted and analyzed. The workflow successfully linked cell surface phenotype, TCR sequence, antigen specificity, and gene expression for individual T-cells, leading to the discovery of novel TCR-antigen interactions.
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