10X Genomics
Performing a 10X Genomics experiment is rewarding but also costly. The adage "Garbage In, Garbage Out" definitely applies; therefore, careful thought should be given to experimental design and sample preparation for your specific research goal. It is important that you contact Core staff for an initial consultation to ensure optimal experimental design and setup. We also highly recommend that you query 10X Genomics resources and contact their technical support team.
10X Genomics Applications
- Single Cell Gene Expression
- Visium Spatial Gene Expression
- Single Cell Multiome ATAC + Gene Expression
- Single Cell Immune Profiling
Single Cell Gene Expression
3' gene expression profiling at scale with single cell resolution.
The Chromium Single Cell Gene Expression Solution provides a scalable microfluidic platform for gene expression profiling of thousands of individual cells per sample. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA molecules from an individual GEM share a common 10x Barcode. Sequencing-ready libraries are generated and sequenced, and the unique 10x barcodes are used to associate reads back to the individual partitions.
Resources
10X Genomics Single Cell Gene Expression Experiment Planning Information
The standard 10x Genomics Single Cell Gene Expression library prep retains mRNAs. lncRNAs and miRNAs are lost.
10X Genomics Resources
There are many resources to be explored and caveats to be made aware of while planning and preparing samples for 10X Genomics Single Cell Gene Expression. We highly recommend querying 10x Genomics resources (https://support.10xgenomics.com/) and contacting their support team while planning your experiment.
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Dr. Brandon Mistretta |
Field Application Specialist |
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Nicky Hales |
Sales Executive (TX/LA/AR/MS) |
Cell Preparation
- Download and read the 10X Genomics Single Cell Preparation Guide (CG000053): https://support.10xgenomics.com/single-cell-vdj/index/doc/demonstrated-protocol-single-cell-protocols-cell-preparation-guide#header
- It is important to contact technical support and inform them of what type of cells and conditions you will be working with, as the recommended cell preparation guidelines may change. Factors such as cell type, tissue type, cell size, viability, etc. will influence cell preparation guidelines.
- For cultured cell lines, 10X Genomics recommends enzymatic dissociation or trypsinization. It is important to optimize the incubation time for your particular cell line, as over incubation may damage cells.
- For primary cells, 10X Genomics has tested FACS (fluorescence-activated cell sorting), magnetic bead purification (e.g., Miltenyi Microbeads), and gradient-based purification (e.g., Percoll, Optiprep, Apheresis) methods.
- Filtering cell suspensions is highly recommended to remove debris and aggregates. Cell strainers with a 40 micron pore size should be used. 10X recommends either the Flomi Cell Strainer or Milteny Biotec MACS SmartStrainer. There are advantages and disadvantages to both. Always do your final cell count after straining, as this process will reduce the number of cells.
- The maximum cell diameter officially supported by 10X Genomics is 30 um, but the maximum theoretical limit is 65 um. These diameters are calculated for cells in suspension, not flattened. If your cells are at the upper limit of this size range, you may want to consider performing a nuclei isolation.
Sample Submission Requirements
- Cells must be fully dissociated and in a single cell suspension.
- Cells should be washed twice and resuspended in 1X PBS + 0.04% BSA. This wash media should be free from calcium, magnesium, and EDTA. If your cells cannot tolerate this solution, 10X genomics support can provide you with an alternative wash media.
- Submit cells in Eppendorf DNA LoBind Tubes or similar (check availability with RCF staff if you are unable to obtain tubes due to COVID ordering restrictions).
- Minimize cell lysis and cell loss. Use wide-bore pipette tips to wash cells. Do not treat cells rough.
- Provide cells at a target concentration of 700 to 1200 cells/ul; do not dilute your entire stock. Provide a minimum volume of 200 ul.
- Please provide Core staff with extra buffer/media to perform cell dilutions if needed.
- Provide cells with viability >70% (with >90% being optimal).
- Minimize the presence of cellular aggregates, dead cells, non-cellular nucleic acids, and reverse transcription inhibitors.
- Bring cells to the Core on ice. Ideally, incubation times should be kept to a minimum (<30 minutes) prior to loading on the Chromium Controller; therefore, coordination with Core staff is critical.
- Cells must be received no later than 2 PM on the scheduled day of submission. You must notify core staff 1 hour prior to cells being delivered, as reagents must be prepared in advance.
- Primary human cells should be screened for infectious agents and that information should be provided to Core staff. Copies of standard operating procedures must be provided prior to submission of cells.
- At least 2 weeks prior to the sample prep, a mock sample prep must be conducted. Samples must be brought to the core. Core staff will count and access cells for viability. This is a requirement to allow time for troubleshooting, resolving discrepancies with cell counts, and conferring with 10x support staff to improve sample prep, if necessary.
Determining Targeted Cell Recovery Amount
- The ideal number of target cells varies greatly with the biological question being asked. Smaller numbers of cells may be sufficient when comparing transcriptional profiles of known cell populations; however, larger numbers of cells may be needed when identifying new or rare subpopulations of cells. If unsure, use at least 3,000 cells as a starting point. 10X Genomics Support can help you determine the ideal targeted cell recovery.
- Approximately 65% of loaded cells will be recovered for library prep and sequencing. You must take this into account when determining if you have ‘enough’ number of cells for this experiment.
Recommended Number of Sequencing Reads
- 10X Genomics recommends a minimum of 20,000 reads per cell. Biological question, cell type, literature may require more. Contact 10X Genomics for assistance in determining the target number of reads.
- If you determine you need more reads for analysis after sequencing, we can sequence the prepared library again, as it is stored in the core freezer.
Analysis
- Core staff do not perform any aspect of analysis for 10X applications.
- Investigators who wish to perform their own analysis can download the software for visualization (Loupe Browser) from 10X Genomics. 10X Genomics Support can assist with data uploading and interpretation of the metrics.
- If you need analysis support, you can reach out to the Center for Applied Immunology and Pathological Process (CAIPP) Modeling Core (Dr. Jian Wang and Dr. Rona Scott). It is advantageous to reach out to them during the planning phase of your 10X Genomics project.
- To download Loupe Browser: https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest?
- Loupe Browser tutorial: https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser
Checklist of information to discuss with 10X Genomics Support:
__ Cell type
__ Cell source (tissue culture, primary cells, etc.)
__ Cell Size
__ Cell viability
__ Cell prep protocol
__ Expecting debris/aggregates? Cell straining, flow, etc.
__ Biological question
__ Targeted cell recovery #
__ Targeted sequencing reads/cell
Checklist of information to Provide Research Core Staff:
Prior to cell prep day:
__ Targeted cell recovery #
__ Targeted sequencing reads/cell
__ What time you expect to have cells isolated and handed off to core staff
Cell prep day:
__ Cell concentration (700-1200 cells/ul)
__ Cell viability (> 70%, ideally >90%)
Costs
Visium Spatial Gene Expression
Simultaneous analysis of molecular and imaging data from tissue sections.
The Visium Spatial Gene Expression Solution measures total mRNA in intact tissue sections and maps the location(s) where gene activity is occurring. Each Visium Spatial or Gateway Gene Expression Slide contains Capture Areas with gene expression spots that include primers required for capture and priming of poly-adenylated mRNA. Tissue sections placed on these Capture Areas are fixed and stained, permeabilized, and cellular mRNA is captured by the primers on the gene expression spots. All the cDNA generated from mRNA captured by primers on a specific spot share a common Spatial Barcode. Libraries are generated from the cDNA and sequenced and the Spatial Barcodes are used to associate the reads back to the tissue section images for spatial gene expression mapping.
Resources
Please explore the 10X Genomics support website for more detailed information. It is advisable that you pay special attention to the sample preparation suggestions.
Fresh-Frozen: https://support.10xgenomics.com/spatial-gene-expression.
FFPE: https://support.10xgenomics.com/spatial-gene-expression-ffpe.
Costs
Single Cell Multiome ATAC + Gene Expression
Simultaneous profiling of 3' gene expression and chromatin accessibility from the same cell.
Chromium Single Cell Multiome ATAC + Gene Expression begins with a single nuclei suspension. Bulk transposition occurs using the enzyme transposase, which preferentially cuts nuclear DNA in open chromatin regions. Transposed nuclei are then partitioned into Gel Beads-in-emulsion (GEM) droplets, with a single Gel Bead containing a unique 10x Barcode. Within the GEM, the unique barcodes are attached to available mRNA and transposed DNA fragments in a single nucleus. Following this incubation, GEMs are broken and pooled before clean up, pre-amplification, and library construction. Two libraries are made from a single pool of GEMs, one for sequencing RNA and one for ATAC.
Resources
Please visit 10X Genomics for resources: https://support.10xgenomics.com/single-cell-multiome-atac-gex.
Sample preparation is key to a successful experiment!
Costs
Single Cell Immune Profiling
V(D)J repertoires of T and B cells integrated with 5' gene expression.
The Chromium Single Cell Immune Profiling Solution provides a scalable microfluidic platform for simultaneous generation of full-length, paired V(D)J sequences, as well as gene expression, cell surface protein expression, and antigen specificity data at a single cell level. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA molecules from an individual GEM share a common 10x Barcode. Sequencing-ready libraries are generated and sequenced, and the unique 10x barcodes are used to associate reads back to the individual partitions. This document provides guidance on how to get started with the Single Cell Immune Profiling assay.
Contact Us
Camille Cannon, MS
Research Specialist, Genomics
camille.cannon@lsuhs.edu
(318) 675-4174
Rona Scott, PhD
Director, CMTV Genomics Core
Professor, Department of Microbiology and Immunology
rona.scott@lsuhs.edu
(318) 675-6263
Paula Polk, MS
Assistant Director, Research Core
paula.polk@lsuhs.edu
(318) 675-4939