10X Genomics

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 Single Cell 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.

Web	https://support.10xgenomics.com/single-cell-gene-expression
Phone	1-800-709-1208
Support Email	Info@10XGenomics.com
Brandon Mistretta, PhD	Field Application Specialist Brandon.Mistretta@10XGenomics.com
Nicky Hales, PhD	Sales Executive (TX/LA/AR/MS) Nicole.Hales@10XGenomics.com

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.

Practice Isolation

A practice isolation with sample imaging for viability and cell count is highly recommended prior to single cell experiment scheduling.  For all practice isolations, the researcher should supply the sample in the appropriate concentration and viability range with minimal debris and clumps.  The investigator should discuss the images with 10X Genomics staff to determine if the quality is appropriate for single cell sequencing.  Core staff can image the samples and send to the researcher.  For core staff imaging, the practice isolation must be coordinated and scheduled in advance, and the researcher will provide Core staff with their cell count and viability calculations.  All troubleshooting should be performed and conditions optimized prior to scheduling the actual experiment.

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 need tubes).
• Minimize cell lysis and cell loss. Use wide-bore pipette tips to wash cells. Do not treat cells roughly.
• 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.
• Make certain cells are at the appropriate dilution prior to providing to RCF staff.
• 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, it is recommended that you conduct a mock sample prep. Samples can be brought to the Core staff if you need a cell count and viability measurement. Doing this in advance will 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’ cells for the experiment.

Sequencing Recommendations

• 10X Genomics recommends a minimum of 20,000 reads per cell.  Biological question, cell type, and literature may suggest you need 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's freezer.

Analysis

• Core staff does not provide any data analysis.  Fastq files will be accessible through Illumina BaseSpace.  Access to the fast files can be shared with any collaborators, as long as the Core receives written permission from the PI.
• The Center for Applied Immunology and Pathological Processes (CAIPP) Modeling Core can perform analysis for a fee.  Please reach out to Drs. Rona Scott (Rona.Scott@lsuhs.edu) or Jian Wang (Jian.Wang@lsuhs.edu)  for more information.  It is advisable that you consult with them during the planning phase of your project.
• If you would like to perform your own analysis, 10X Genomics provides free resources.  You can download Loupe browser from their website at: https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest?  The Loupe browser tutorial can be accessed at: 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%)

Application	Internal Fee	External Fee
Single-Cell RNA Sequencing, Manual	$150/sample	$250/sample

This assay investigates mRNA expression in formalin fixed & paraffin embedded (FFPE) tissue by using probes against either the human or mouse transcriptome. Gene expression is spatially mapped. Since this assay utilizes poly(A) capture, only mRNA information will be retained. It is possible to obtain information from viruses only if they are poly(A) viruses. Each Visium Spatial FFPE Gene Expression Slide has either 2 or 4 capture areas, measuring 6.5 x 6.5 mm. Each capture area has ~5,000 gene expression spots. Each barcoded expression spot is 50 um, meaning it is possible that you will not decipher each individual cell’s gene expression.

Investigators are responsible for tissue sectioning, tissue placement on the Visium slide, deparaffinization, and staining prior to delivering the Visium slide to the Research Core Facility.

You MUST coordinate Visium processing dates with Core staff prior to placement of tissue on the slide. The Core WILL NOT accept any samples that have not been scheduled in advance.  Keep in mind that Core staff can only process one slide at a time. 

10X Visium Genomics Resources

There are many resources to be explored and caveats to be made aware of while planning and before preparing samples for 10X Genomics Visium applications. Please contact the 10X support team and inform them of what type of tissue and conditions you will be working with, as the recommended preparation guidelines may change.  Also, we recommend exploring the 10X Genomics support website for more detailed information.  It is advisable that you pay special attention to sample preparation suggestions.  

Web	https://www.10xgenomics.com/support/spatial-gene-expression-ffpe https://www.10xgenomics.com/support/spatial-gene-expression-fresh-frozen
Phone	1-800-709-1208
Support Email	Info@10XGenomics.com
Mahdis Rahmani, PhD	Visium Field Application Specialist Mahdis.Rahmani@10xgenomics.com
Nicky Hales, PhD	Sales Executive (TX/LA/AR/MS) Nicole.Hales@10XGenomics.com

Assessing RNA Quality

• It is highly recommended that you assess RNA quality of the FFPE tissue block prior to tissue mounting by isolating RNA with the recommended Qiagen kit. Core staff can assess the quality of the RNA on an Agilent TapeStation. It is recommended to proceed with Visium only if > 50% of RNA is 200 bp or larger, but 10x Genomics has seen good quality experiments with only 28% RNA 200 bp or larger.

Tissue Preparation and Placement

Please download and read the most recent guides at https://www.10xgenomics.com/support/spatial-gene-expression-ffpe.  Make certain that you download the guides that fit your workflow (i.e. standard tissue placement vs. CytAssist, H&E vs. Immunofluorescence staining and imaging).  If you are not sure which guides are appropriate, please ask Core staff.  It is also useful to review the Visium Spatial Gene Expression for FFPE Protocol Planner at https://support.10xgenomics.com/spatial-gene-expression-ffpe/index/doc/user-guide-visium-spatial-gene-expression-for-ffpe-protocol-planner.

• The recommended thickness for most tissue is 10 um.
• It is important that you practice tissue placement on a plain glass slide prior to the experiment.  Print the Visium Spatial Layout template located on page 26 of the Visium Spatial Gene Expression for FFPE Tissue Preparation Guide (CG000408).  Draw the frames on the back of the blank slide and practice tissue placement in the squares.  Alternatively, you can purchase practice slides with fiducial frames from 10X (Visium Tissue Section Test Slide, PN 2000460).  This is also a good time to optimize staining.

• Handle Visium slides carefully so as not to damage fiducial frames, probes, or the special label containing a serial number. Do not wipe or attempt to clean slides.

• Store unused Visium slides in a dry environment at room temperature in their original packing. Do not remove desiccant.

• The tissue should be placed inside the fiducial frame: no data will be generated for tissue on or outside of the frame. The fiducial frame is used to map the slide, so only minimal tissue covering of the fiducial frame is acceptable.

• It is important that your tissue is placed evenly on the slide.  A loss of spatiality will occur for tissue areas that have folds/wrinkles or detach from the slide.

Deparaffinization and Staining

• Deparaffinization and staining must be performed by the investigator.  Imaging can be done by core staff but this must be requested when scheduling your appointment. Decrosslinking, and all other downstream procedures, must be done by Core staff. 

• Refer to one of the two guides below for assistance with deparaffinization and staining:

  H&E	10X Demonstrated Protocol CG000409
  Immunofluorescence	10X Demonstrated Protocol CG00041

• Note that the Visium Cassette mentioned in these protocols is only necessary for downstream library prep, hence, investigators do not need to handle the Visium cassette.

• Tissue-mounted Visium slides should be incubated at 42°C for 3h and dried overnight at room temperature in a desiccator, prior to deparaffinization. According to 10X Genomics, as a safe stopping point, tissue-mounted Visium Slides can be stored for up to 2 weeks at room temperature in a desiccator prior to deparaffinization.

• The core has a thermocycler adaptor that can be borrowed for the desiccation & deparaffinization step, if the investigator choses to not use a dryer oven. This thermocycler adaptor must be returned at time of Visium processing, as it is required for library preparation.

Imaging

• Core staff can image the slide at up to 40x with an EVOS M7000 microscope at no additional cost. This microscope is equipped for brightfield microscopy as well as some fluorescence channels (GFP, RFP, Cy5, DAPI).
• Optionally, investigators can image the slide with their own system or with a VS200 SlideView slide scanner (Olympus Life Science) located in the Tissue and Serum Repository.  The cost is $50 an hour; please reach out to Dr. Ellen Friday (ellen.friday@lsuhs.edu) for use.
• Investigators who wish to image themselves should consult 10x Genomics for imaging requirements. Download and review the Visium Spatial Gene Expression Imaging Guidelines (CG000241), if you prefer to image yourself.
• If imaging is not performed by core staff, the stained and imaged slide will be transferred to core staff on the scheduled day.

Sample Submission Requirements

• The stained, tissue-mounted Visium slide should be transferred to Core staff the same day as deparaffinization and staining.  This day will have been scheduled in advance.
• The slide must be provided to the Core no later than 2PM on the scheduled day of submission.  
• You must notify Core staff 1 hour prior to delivering the slide, as the microscope and reagents must be prepared in advance.
• Core staff can only process one slide at a time; therefore, investigators cannot deparafiinize and stain multiple slides in one day for the Core to process.

Sequencing Recommendations

• 10X Genomics recommends a minimum of 25,000 read pairs per tissue covered spot on the capture area.  Biological question, cell type, and literature may suggest you need more.  Contact 10X Genomics for assistance in determining the target number of reads.
•  It is recommended that you do not purchase the Illumina sequencing kit until the estimated coverage area is calculated.  For example, if your tissue is only covering ~50% of the capture area, you do not need as large a sequencing kit as if you were covering 100% of the area.
• If you determine you need more reads for analysis after sequencing, the Core can sequence the prepared library again.

Analysis

• Core staff does not provide any data analysis.  Fastq files will be accessible through Illumina BaseSpace.  Access to the fast files can be shared with any collaborators, as long as the Core receives written permission from the PI.
• The Center for Applied Immunology and Pathological Processes (CAIPP) Modeling Core can perform analysis for a fee.  Please reach out to Drs. Rona Scott (Rona.Scott@lsuhs.edu) or Jian Wang (Jian.Wang@lsuhs.edu)  for more information.  It is advisable that you consult with them during the planning phase of your project.

Checklist of information to discuss with 10X Genomics Support:

__        Tissue type (some tissues impact detachment)
__        Species (human or mouse)
__        Treatment (some treatments impact detachment)
__        Replicates recommended (Usually recommend between 2-4)
__        Biological question
__        Targeted sequencing reads/spot
__        Practice slide staining & imaging
__        Tissue placement, folding/wrinkles, detachment issues
__        Imaging requirements, if investigator images slides

Checklist of information to Provide Research Core Staff Prior to experiment day:

__        Species (Human or Mouse)
__        Imaging plan (who is imaging, H&E or fluorescent staining)
__        Practice slide staining & imaging
__        How many slides total (core staff can only process one slide at a time)
__        Schedule Visium experiment (Date and time)

Visium FFPE Timeline:

• Practice placing tissue on a regular slide
• Practice staining & imaging tissue. Send images to 10x to determine quality of tissue placement/staining/imaging
• Do not mount tissue on Visium slide until coordination with the core for processing:
  • Remember, the core can only accept one Visium slide at a time for processing.
  • Tissue can be mounted on the Visium slide up to 2 weeks before scheduled processing.
  • The day of scheduled processing with the core, the slide must be deparaffinized, stained, and imaged.
  • The slide must be received in the core no later than 2PM on the scheduled day of submission.

Application	Internal Fee	External Fee
Visium FFPE	$200/sample	$325/sample
Visium Fresh Frozen	$200/sample	$325/sample
Tissue Optimization for Fresh Frozen	$150/tissue slide	$250/tissue slide

Please visit 10X Genomics for resources:  https://support.10xgenomics.com/single-cell-multiome-atac-gex.

Sample preparation is key to a successful experiment!

 

 

Application	Internal Fee	External Fee
Single-Cell Multiome ATAC + Gene Expresson	$300/sample	$500/sample
Upload to 10X Cloud for Analysis	$150/upload	$250/upload