This webinar, presented by José M. Morachis (NanoCellect Biomedical) and Dr. Brian R. Fritz (10x Genomics), focuses on optimizing sample preparation for 10x Genomics single-cell applications through microfluidic-based cell sorting. The first half, led by José Morachis, introduces the critical importance of isolating target cell populations and removing impurities (debris, dead cells, doublets) for high-quality single-cell sequencing data, emphasizing the gentle sorting capabilities and user-friendliness of the NanoCellect Wolf Cell Sorter. He provides case studies illustrating improved viability, purity, and data quality (e.g., more distinct cell clusters, higher gene counts) in various cell types and even plant nuclei when using upstream cell sorting. The second half, by Dr. Fritz, delves into 10x Genomics' multi-omic cytometry, showcasing how their platform enables simultaneous analysis of RNA, protein expression, T-cell receptor sequences, and antigen specificities from single cells. He highlights the utility of combining flow cytometry with 10x Genomics for resolving rare cell populations and complex biological states, demonstrating comprehensive immune profiling applications and the discovery of novel T-cell receptor specificities.
Optimizing Single-Cell Genomics: The Power of Upstream Cell Sorting and Multi-Omic Analysis
0:09 Webinar Introduction: Susie Valdez of LabRoots introduces the webinar, sponsored by NanoCellect Biomedical and 10x Genomics, focusing on microfluidic-based cell sorting for 10x Genomics applications.
2:31 The Importance of Single-Cell Analysis: José Morachis emphasizes the need to understand individual cells to uncover their "secrets" in various applications like genomics, gene editing, and cell therapy, moving beyond population-level analysis.
3:58 Sample Preparation Challenges: For workflows like 10x Genomics, sample quality is critical. Issues like cell debris, dead cells, and aggregates can lead to clogged systems, degraded RNA, and reduced sequencing accuracy, wasting resources.
5:02 Importance of Target Cell Isolation: Proper sample preparation involves isolating target cells and removing background material to ensure unbiased and accurate scientific results.
5:37 Gentle Cell Handling Tips: General tips for maintaining cell viability include using wide-bore pipette tips, avoiding excessive vortexing, gentle centrifugation, and optimizing buffers/temperatures.
6:31 Benefits of Enrichment & Cleanup: Enriching target cells while removing debris, dead cells, and aggregates prevents clogging, preserves RNA integrity, and improves sequencing accuracy.
7:21 Cell Isolation Techniques Overview: Common techniques include magnetic bead cell sorting (simple, limited) and fluorescence-activated cell sorting (FACS) (high-speed, multi-parameter, but complex/less accessible).
8:20 NanoCellect Wolf Cell Sorter: Introduction of the Wolf Cell Sorter as an easy-to-use, gentle (low pressure, <2 PSI vs. 55-70 PSI for traditional sorters), small-footprint instrument. It ensures high cell viability and uses a sterile, disposable microfluidic cartridge to prevent cross-contamination.
11:17 Practicality of Upstream Sorting: Illustrates how sorting upstream significantly increases the percentage of useful cells for analysis, leading to more cost-effective experiments and better data quality (e.g., 50% vs. nearly 100% useful data).
12:48 CHO Cell Example: Demonstrates how sorting CHO cells with the Wolf sorter removes dead cells and debris, yielding a much cleaner, viable cell population for downstream analysis.
13:44 T-Cell Sorting Example: Shows improved viability and purity for CD3+ T-cells sorted from PBMCs, allowing for more detailed analysis of T-cell subsets with 10x Genomics. Sorted cells maintain functionality.
15:13 Multi-Parameter Barcoding Technologies: Highlights new applications using barcode technologies from BioLegend (TotalSeq) and Immudex (Dextramer) for multi-parameter analysis with 10x Genomics.
16:01 Rare Population Enrichment: Explains how pre-enrichment (e.g., using Akadeum beads for CD4+ cells) followed by Wolf sorting can efficiently isolate rare cell populations (e.g., regulatory T-cells), achieving >90% purity consistently.
17:43 Nuclei Isolation from Tissue: Addresses challenges with obtaining single-cell suspensions from difficult tissues (e.g., brain, plants) by isolating and analyzing nuclei, which can provide comparable insights. Example shown with tomato and bell pepper nuclei.
20:07 10x Genomics Analysis Comparison (Sorted vs. Unsorted PBMCs): Direct comparison shows that sorted PBMCs yield improved median genes per cell, lower percent reads not associated with a cell, and higher median UMI counts. Sorted samples also show more distinct clusters (9 vs. 7).
21:56 Stem Cell Sorting Comparison: Studies with sensitive iPS cells show that both Wolf sorting and traditional FACS (BD FACS Aria) improve data quality (median genes, UMI counts) compared to unsorted, with Wolf sorting yielding even better results.
23:17 Importance of Optimization: References a paper highlighting that downstream analysis results are highly dependent on meticulous sample optimization and handling.
25:52 Brian Fritz - Multi-Omic Cytometry Introduction: Brian Fritz transitions to discussing multi-omic cytometry, specifically immune profiling, and its application in understanding biological complexity.
26:38 Multi-Omic Measurement Types: Details various analytes that can be measured simultaneously from single cells using 10x Genomics: RNA, proteins, targeted gene expression (T/B cell receptor genes), antigen specificities, and potentially chromatin states or CRISPR guide RNAs.
29:40 10x Genomics Workflow: Explains the microfluidic system that partitions single cells with barcoded primer libraries (GEMs – Gel Bead in Emulsion) for reverse transcription and subsequent bulk library prep. Up to 80,000 cells per chip, 8 samples in parallel.
31:52 Gel Bead Oligonucleotide Technology: Describes how gel beads are coated with oligonucleotides containing unique molecular identifiers (UMIs) for transcript counting and 10x barcode sequences for assigning transcripts to individual cells.
33:40 Antibody & Dextramer Integration: Specific oligonucleotide sequences on the gel bead allow capture of complementary barcodes conjugated to antibodies (for protein epitope mapping) and Immudex Dextramers (for antigen specificity), enabling multi-omic analysis.
35:16 CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing): Discusses the foundational work combining flow cytometry's antibody specificity with next-generation sequencing, allowing simultaneous RNA and protein analysis.
36:11 Multi-Omic Cytometry Examples: Demonstrates how 10x Genomics can separate T-cells based on CD4/CD8 expression (similar to flow cytometry) but with the added power of mapping gene expression states onto protein profiles.
38:00 T-Cell Receptor (TCR) Analysis & Antigen Specificity: Explores advanced applications, including simultaneously analyzing full-length TCR alpha/beta/gamma/delta genes, cell surface epitopes, and antigen specificities using Dextramer reagents.
40:08 Reagent Quality Control (QC): Emphasizes the importance of QC for antibody and Dextramer reagents, comparing flow cytometry and 10x Genomics results to ensure concordant and optimal staining.
43:20 Comprehensive Immune Profiling Workflow: Detailed protocol for sorting CD8+ and Dextramer+ T-cells from PBMCs, integrating dead cell removal and subsequent 10x Genomics analysis to capture gene expression, protein expression, TCR sequences, and antigen specificity from the same single cells.
45:51 Large-Scale Application & Novel Discoveries: Describes inputting millions of CD8+ T-cells and multiplexing 50 Dextramer molecules to identify thousands of antigen-binding cells, leading to the discovery of 25 novel TCR antigen specificities added to databases.
48:15 Summary & Call to Action: Reaffirms the power of combining flow cytometry with multi-omics for rare cell identification, heterogeneity definition, and a holistic workflow from sample acquisition to bioinformatics. Encourages audience to reach out for more information.
49:52 Q&A: RNA vs. Protein Discrepancy: Brian Fritz explains that discrepancies between RNA and protein detection are common due to different half-lives and regulatory mechanisms, providing deeper insights into gene expression dynamics.
51:12 Q&A: Epithelial Cell Sorting & Input Requirements: Jose Morachis confirms that epithelial cells can be sorted, emphasizing the need for single-cell suspension and a minimum of 10,000-20,000 target cells, though 10x Genomics often recommends more.
52:32 Q&A: Wolf Sorter Processing Speed: The Wolf sorter can process about 1 mL of sample in 40 minutes, with setup, totaling about an hour for a full sample, depending on concentration (100,000 to 1 million cells/mL for higher purity).
53:44 Q&A: 10x Genomics Gene Detection: Brian Fritz states that typically thousands of genes per cell can be detected, varying by cell and sample type, with more transcriptionally active cells yielding higher numbers.
55:18 Q&A: Tissue Sample Processing: Brian Fritz notes that many 10x Genomics applications use tissue as a starting sample, with protocols available online. Nuclei isolation can be an easier alternative for difficult tissues.
56:26 Q&A: Multi-Omics Parameters: Brian Fritz indicates that the 10x multi-omics cytometry workflow can analyze well over a hundred (even 300+) cell surface epitope parameters simultaneously, vastly exceeding traditional cytometry.
57:41 Q&A: COVID-19 Data: Brian Fritz confirms 10x Genomics has a COVID-19 resource page with numerous customer publications and pre-prints, though they don't handle BSL3 samples internally.
58:38 Closing Remarks: Both speakers thank the audience and invite further individual discussions for specific workflow solutions.