10X Genomics FAQs

Q: What factors should I consider when planning my antigen specificity experiment?

A: Our compatible partner Immudex offers oligo-barcoded dextramer reagents ( the dCODE Dextramer ) for determining antigen specificity. This product consists of a dextran backbone with a series of antigen-presenting MHC molecules (for interacting with T-cell receptors), a fluorescent molecule (Phycoerythrin), and a DNA barcode that is compatible with our Immune Profiling Solution (see below):

[email protected]

Thisallows for the detection of antigen-specific T-cells through use with our Feature Barcoding technology. The general workflow for using dextramer reagents will consist of obtaining a single-cell suspension, labeling the suspension with the dextramers and antibodies of interest, washing the suspension, then flow-sorting for dextramer specific T-cells (optional) before proceeding immediately to the correct Immune Profiling Solution User Guide with Feature Barcoding.

Below are some factors that should be considered when planning an experiment with dextramers:

What percentage of the cell suspension is interacting with my antigen(s) of interest?The percentage of T-cells in a diverse cell population that interacts with a specific antigen is likely to be very low (frequently less than 1%). Therefore, when looking for antigen-specific interactions, it is recommended to sort for both dextramer positive interactions and also T-cell markers in order to increase the signal and reduce background. Please see this figure from our application note 'Redefining Cellular Phenotyping: Comprehensive Characterization and Resolution of the Antigen-Specific T Cell Response' as an example:

In the above figure, you can see that it is difficult to detect specific antigen-TCR interactions in the unsorted samples; when sorting the cells for T-cell markers and dextramer-positive interactions, we see a much stronger signal.

Do I plan to perform antibody labeling in addition to dextramer labeling (either for flow sorting or for use with Feature Barcoding technology)?It is entirely feasible to stain cells with both dextramers and antibodies, but a major workflow consideration to take into account is that dextramer staining needs to be performed first. Dextramers do not bind as strongly as antibodies, and thus staining with antibodies first or at the same time can lead to crowding out of the dextramer interaction with the TCR. Please see our Demonstrated Protocol on Cell Labeling with Dextramer Reagents for more information.

Experience level with flow sorting:Flow sorting is an advanced sample preparation method. Therefore, we recommend that customers interested in performing this technique either have previous experience with flow sorting or have access to resources (such as a sorting facility) who can help with this procedure in order to increase the chances of success.

Our Demonstrated Protocol on Cell Labeling with Dextramer Reagents provides an example gating strategy for flow sorting. Please keep in mind that these are recommendations and may not be applicable for all sample types/experiments.

Compatibility of the MHC allele to target sample:Dextramer MHC allele and donor allele should match

Controls:Some level of background staining is common when working with dextramers, so having a negative control for comparison purposes is very important. Immudex support can offer more guidance on the appropriate controls to select for your antigen of interest.

For additional questions on dextramers, we recommend reaching out tofor more information.

Question:Is it possible to interpret similarities between gene expression in clusters based on distance from one cluster center to another cluster center in the 2D t-SNE space? If not, is there another way to use the 10x single-cell data to say that two clusters are more similar to each other than they are to the rest of the clusters?

Answer:We recommend against using the distances between the clusters in t-SNE space to interpret how similar any two particular clusters are.The distances between points/cells are relative - because the algorithm is non-linear and adapts to underlying data, the distances on the x and y-axis have no direct interpretation. As described in this t-SNE primer, "The algorithm is non-linear and adapts to the underlying data, performing different transformations on different regions."If you want to evaluate differences between any two clusters, you could run a differential expression analysis between the clusters to identify the differentially expressed genes. You can do this in Loupe Cell Browser. Here is some more information on how to compute differential expression between cell clusters.

Prior to t-SNE, the normalized gene-barcode matrix is reduced from having dimensions (number of genes x number of barcodes) to (top 10 principal components x number of barcodes) via principal components analysis. In turn, the top 10 PCs are used as inputs to both t-SNE and the various clustering approaches (graph-based or k-means). The t-SNE visualization and clustering/cluster assignment are independent steps/approaches. Thus you may see some cells assigned to a cluster that overlap or co-localize with cells in another cluster in the t-SNE space.For more information on t-SNE, please refer to this publication:Maaten, L.V.D. and Hinton, G., 2008.Visualizing data using t-SNE. Journal of Machine Learning Research, Vol 9(Nov), pp. 25792605.

Question: How to resolvemkref error: BUG: next index is smaller than previous, EXITING?

Answer: The error above is specific to the genome indexing step using STAR genomeGenerate.

It has been observed when using an input genome FASTA file with repeat-masked sequences and resolved when an unmasked version of the FASTA file is used instead.

For example, the error was observed when using the repeat-masked version of the pig genome assembly from Ensembl (Sus_scrofa.Sscrofa11.1.dna_rm.toplevel.fa) and resolved with the use of the unmasked version (Sus_scrofa.Sscrofa11.1.dna.toplevel.fa).

Question:What is the difference between the filtered and the raw (unfiltered) feature-barcode matrix?

Answer: The raw gene-barcode matrix includes all valid barcodes from GEMs (Gel Bead-In EMulsions)captured in the data. However, because most GEMs do not actually contain cells, it follows that most barcodes in the data do not correspond to cells, but rather background noise (e.g. GEMs with free-floating mRNA from lysed or dead cells).

The filtered gene-barcode matrix excludes barcodes that correspond to this background noise. This can be visualized in the barcode vs UMI count rank plot in the web summary file. In the example plot below, UMI counts are on the y-axis ranging from 0 to 10,000 in log scale. Barcodes are on the x-axis, ranked from 0 to 1,000,000 also in log scale. Cell-associated barcodes as determined by the cell-calling heuristic are in blue whilebackground barcodes are in gray.

algorithms overview page

For more information on gene-barcode matrices please see this page.For more information on the cell-calling heuristic, please see the "Calling Cell Barcodes" section of the .

Question: I am trying to get the B Cell isotype information for the annotated contigs, where can I find this information?

Answer:B cell isotype information can be obtained from various output files generated by "cellranger vdj" pipeline.

1) From all_contig_annotations.csv file. This file is described in detail here. One of the columns in this file "c_gene" identify the C gene assigned for a given contig. The C gene in this column can identify the isotype of the contig.

2) You can also get this information interactively by opening the .vloupe file in Loupe V(D)J Browser.

When viewing a B cell sample, there are two additional summary charts which allow you to analyze the isotype distribution. To access these, return to the chart view by clicking the bar chart icon. Then click theChart Typedropdown and scroll to the bottom of the list to find theB-Cell IsotypesandB-Cell Paired Isotypescharts.

First, select theB-Cell Isotypechart. This chart displays the frequency of immunoglobin isotypes and type of light chains in the sample.

the Loupe V(D)J Browser Tutorial: B Cells

Next, select theB-Cell Paired Isotypechart. The chart displays the distribution of paired isotypes and light chains among clonotypes in the sample. Only clonotypes where there is exactly one heavy chain and one light chain are included in this chart.

For more information on working with B cell data in Loupe V(D)J Browser, please see.

Question: When I try to run "cellranger aggr" pipeline with >128 k cells I get an error. Is there an upper limit on the number of cells that can be used with "cellranger aggr" pipeline?

Answer:Cell ranger does not have a maximum number of cells threshold when "aggr" is run without batch correction. We have validated for up to 250k cells when run on minimum compute resources. But if you have larger compute resources, you could aggr larger cell counts in Cell Ranger 3 when merging data without chemistry correction.However, if you are using chemistry batch correction, then there indeed is a threshold of 128k cells. The chemistry batch correction is resource intensive and so considering the minimum compute resources (64 GB RAM), we have specified the 128k cell limit. If however you have sufficiently high compute resources, you can try with manually changing the limit that is hardcoded here:

cellranger-cs/3.x.x/lib/python/cellranger/analysis/constants.pyCBC_MAX_NCELLS =128000Please note that we have not validated Cell Ranger with cell counts beyond this number and advise caution.

Also, please note that Loupe Cell Browser can load up to 1.3 million cells at this time. In addition, the differential expression analysis in Loupe Cell Browser can be done on 100k cells only.

Disclaimer:This article and code-snippet are provided for instructional purposes only. 10x Genomics does not support or guarantee modifications to Cell Ranger code base.

Question:What genes should I filter using the mkgtf tool when making a custom reference for Cell Ranger?

Answer: In order to create a custom reference, you will start with a GTF file that contains gene annotations. We recommend filtering the GTF file so that it contains only gene categories of interest by usingcellranger mkgtftool.Which genes to filter depends on your research question. The attributes used for filtering in pre-built 10x references include:

Protein-coding genes (--attribute=gene_biotype:protein_coding)

Long intergenic noncoding RNAs (--attribute=gene_biotype:lincRNA)

Antisense (--attribute=gene_biotype:antisense)

Psuedogenes (--attribute=gene_biotype:pseudogene)

V(D)J germlinegenes, for example:

--attribute=gene_biotype:IG_V_gene

--attribute=gene_biotype:TR_V_gene

Of note is that Cell Ranger does not use reads that map to multiple genes towards expression (UMI) counting. Therefore having overlapping genes in your reference will lead to reads being ignored. From this perspective, your reference should have only a small number of overlapping gene annotations.

For more information please see Using Custom References.

Question: How do I specify memory and cores for Supernova?

Answer: Please use the --localmemand/or --localcores options when invoking asupernova run. These options should be thought of as throttles rather than hard limits. If you are submitting a job to a cluster or cloud, you may need to request memory/cores in your shell script or qsub command equal to or greater than what you specify with the --localmem and --localcores option. The details of how to do this will vary based on your cluster setup. For more information please see our System Requirements and Troubleshooting Supernova pages, and please direct any questions to [email protected].

Question:How is "Sequencing Saturation"calculated?

Answer:The web_summary.html output from cellranger count includes a metric called "Sequencing Saturation". This metric quantifies the fraction of reads originating from an already-observed UMI. More specifically, this is the fraction of confidently mapped, valid cell-barcode, valid UMI reads that are non-unique (match an existing cell-barcode, UMI, gene combination).

The formula for calculating this metric is as follows:

Sequencing Saturation = 1 - (n_deduped_reads / n_reads)

where

n_deduped_reads = Number of unique (valid cell-barcode, valid UMI, gene) combinationsamong confidently mapped reads.

n_reads = Total number of confidently mapped, valid cell-barcode, valid UMI reads.

Note that the numerator of the fraction isn_deduped_reads, not the non-unique reads that are mentioned in the definition.n_deduped_readsis a degree of uniqueness, not a degree of duplication/saturation. Therefore we take the complement of(n_deduped_reads / n_reads) to measure saturation.

Question: Can we use the cellranger vdj pipeline on organisms that do not have a referencegenome?

Answer: Yes. The cellranger vdj pipeline will work with organisms that do not have a reference genome or have a reference genome with poorly annotated V(D)J genes. In these cases, youcan use the --denovo mode to turn off thereference specific stages (read filtering and V(D)J annotation).

When run in --denovo mode, the pipeline will still assemble contigs de novo. When running in --denovo mode .vloupe files for importing the data to Loupe V(D)J Browser are not generated. Please see this page for more details.

Question: Is there an obvious way of telling which barcodes contain multiple cells (multiplets) for V(D)J libraries?

Answer:At this time we do not have a good way of distinguishing GEMs with multiple cells. Currently we estimate that the doublet rate for VDJ libraries should be the same as GEX libraries.

It is expected that each cell-barcode typically contains one productive TRA and one productive TRB contig. Barcodes with multiple alpha/beta chains (in case of TCR) might throw some light onto the GEMs with more than 1 cells.

If multiple cells with the same clonotype have more than one alpha/beta chains, they are unlikely to be true doublets. The chance of multiple GEMs containing identical multiple chains is very low. If only one cell has multiple alpha or beta chains, it is hard to tell if it is a doublet versus a true biological phenomenon. The rate of true biological double-TRB or double-TRA cells is low. So these barcodes could be a doublet, a barcode collision event, or a PCR chimera. But there is no way to tell with 100% certainty from this data.

Question:Are there methods for identifying multiplets or doublets in single-cell gene expression analysis?

Answer:At present, there is no explicit support for multiplet identification in the Cell Ranger gene expression analysis pipeline other than for the analysis of "barnyard" or mixed-species datasets mapped to a multiple-species reference.

Below is a listing of some third-party tools for detecting doublets/multiplets in single-cell gene expression datasets:

Scrublet:

https://www.biorxiv.org/content/early/2018/07/09/357368

https://github.com/AllonKleinLab/scrublet

DoubletDecon:

https://www.biorxiv.org/content/early/2018/07/08/364810

https://github.com/EDePasquale/DoubletDecon

DoubletFinder:

https://www.biorxiv.org/content/early/2018/06/26/352484

https://github.com/chris-mcginnis-ucsf/DoubletFinder

Please contact the respective developers of each tool for more information.

Disclaimer:This article and code-snippet are provided for instructional purposes only. 10x Genomics does not support or guarantee the code.

Question:Why does the Gene Table in Loupe Browser highlight up-regulated genes by default?

Answer: Up-regulated genes are more straightforward to interpret than down-regulated genes when analyzing single-cell gene expression data.

Since only a fraction of expressed transcripts can be detected in most single gene expression assays, a gene may have zero or low UMI counts for reasons other than biology i.e. the absence of evidence is not evidence of absence.

Displaying up-regulated genes is also more consistent with how clusters aretypically annotated (using enriched expression or up-regulation of known cell-type specific markers).

Question:Does cellranger count preserve the STAR alignment log output(Log.out) files?

Answer:The STAR output logs are not preserved by cellranger count.

The Cell Ranger pipeline splits the initial input FASTQ files into chunks. STAR runs on each chunk separately and generates a log file for each chunk. The intermediate outputs from these chunks, including the STAR logs, are removed by the pipeline to save disk space.

Summary information such as the genomic distribution of mapped reads (intergenic, intronic, exonic and antisense) is provided in the web summary file.Alternatively, the Cell Ranger BAM output file can be queried directly or summarized based on the FLAG, MAPQ, and a variety of other features described on our support site here.

Question: I gotan error "Empty barcode list supplied" when importing a category CSV file into Loupe Cell Browser. The barcodes are correct.

Answer: This error is typically associated with hidden characters such as carriage returns in the CSV file. Loupe Cell Browser requires that files are in plain CSV format.

If you are working in Excel, please ensure that you save the file in plain CSV format.

To remove carriage returns on the Bash shell, please try:

tr '\r' '\n' < category.csv > category.fixed.csv

In general, the solution is to replace each carriage return (\r or ^M) character with a Unix new-line character (\n).

Question:Why can't I find cellranger mat2csv?

Answer: cellrangermat2csv was added in Cell Ranger version 2+ and is not available in earlier versions. It is a utility for converting the sparse gene-barcode matrix files into a dense matrix format CSV file.

To download the latest version of Cell Ranger please see this page.

For more information on mat2csv, please see the section " Converting to CSV Format."

Question: Are 10x Single Cell gene expression libraries strand-specific?

Answer: Yes, 10x Single Cell solutions are strand-specific. Cell Ranger 'Count' counts sense-strand reads only. There is a possibility of legitimate antisense transcripts but they are unlikely to follow the same exon structure as the sense transcripts. We do include a measurement of the levels of antisense transcripts detected in Cell Ranger, which typically hovers around 1% of mapped reads.Those antisense transcripts won't contribute any UMI counts since Cell Ranger does not count UMIs from antisense reads.

Products: Single Cell Gene Expression, SIngle Cell Immune Profiling

Question:Can library and gel bead kits be used past their expiration dates?

Answer: We cannot guarantee performance of reagents if they are used past their expiration date.

To ensure best results, reagents should be used before expiration date and the following considerations should be kept in mind:

Check number of freeze-thaw cycles for the Gel Beads and all kit components stored at -20C (Modules 1 & 2).Avoid more than 10 freeze-thaw cycles.

Unused Gel Beads should be stored at 80C.

The resuspended v2 RT-Primer (PN-310354) and v3Template Switch Oligo (PN-3000228) should be stored at80C.

All enzyme components and Master Mixes should be kept on ice during setup and promptly moved to the recommended storage temperature as soon aspossible.

Check ligase buffer and Additive A for precipitation after thawing and make sure they are fully resuspended before using. If remaining crystals are visible, briefly warming up the solution will facilitate resuspension. Additive A should have a strong smell. If it does not, do not use.

Products:All

Question:Why is my total cDNA yield low?

Answer:Total cDNA yield is highly dependent on the cell type (RNA amount per cell; see notes below), cellpreparation, and the cell number (note that the number of PCR cycles for cDNA amplificationisadjusted as a function of the targeted cell number).

Causes:

Low cDNA recovery may be the result of:

Low total mRNA contentper cell (cell-type dependent)

Loading fewer cells than expected (dependent on cell counting technique and accuracy)

Conditions used to pre-process cells (dissociation, FACS, any conditions that cause lysis, etc.)

Cell viability (low cell viability can reduce transcriptional output as cells enter apoptosis)

RT inhibitory contaminants (carryoveradditives such as EDTA or Mg2+, or RNases/proteases from the cell suspension buffer/media)

Sample clog

Loss of cDNAduring the emulsion breaking orclean-up steps

Resuspension of TSO in the incorrect buffer (regular TE instead of low TE, for example)

Recommendations:

If you are consistently experiencing low cDNA yields and suspect your samples have low RNA content (small cells, nuclei), experiment with adding an extra 1-2 PCR cycles in the cDNA amplification and/or Sample Index PCR steps. However, keep in mind that the extra PCR cycles may increase PCR artifacts and may not increase final library complexity.

Notes:

Primary cells and cells of small cell size (i.e. PBMCs) generally have lower RNA content compared to immortalized cell lines or cancer cells.

We have successfully generated libraries from cDNA yields as low as~1-2 ng.

A typical cDNA yield of 1000 RNA-poor cells is <5 ng/ul

A typical cDNA yield of 1000 RNA-rich cells is >10 ng/ul

Products:Single Cell Gene Expression, Single Cell Immune Profiling

Question:Is the i7 Sample Index plate compatible across products?

Answer:It depends on the product.

Single Cell 3' v2 and v3, Single Cell V(D)J 5', and Genome/Exome products use the same Chromium i7 Multiplex Kit (PN-220103).

Single Cell ATAC uses the Chromium i7 Multiplex Kit N (PN-1000084). These sample indexes are not compatible with other kits.

Single Cell 3' v1 uses a separate set of sample indexes. These sample indexes are not compatible with otherkits.

The sequences of the sample indexes for each kit can be found on our website:

For Genome/Exome Libraries: https://support.10xgenomics.com/genome-exome/index/doc/specifications-sample-index-sets-for-genome-and-exome

For Single Cell 3' Libraries: https://support.10xgenomics.com/single-cell-gene-expression/index/doc/specifications-sample-index-sets-for-single-cell-3

For Single Cell V(D)J Libraries: https://support.10xgenomics.com/single-cell-vdj/sequencing/doc/specifications-sample-index-sets-for-single-cell-vdj

For Single Cell ATAC Libraries: https://support.10xgenomics.com/single-cell-atac/sequencing/doc/specifications-sample-index-sets-for-single-cell-atac

Products:Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell ATAC, Genome/Exome, Single Cell CNV

Question:If my clonotype has multiple alpha and beta chains (for example 1 alpha chain and 2 beta chains), what do I do? Should I discard the clonotype? Or maybe just pick one of the chains?

Answer: Biologically, some fraction of T cells are known to express 2 alpha + 1 beta or 1 alpha + 2 beta chains ( Ref1, Ref2 ). Similarly, there is evidence that some B cells express more than 1 heavy or light chains ( Ref3 ).

Therefore, if you find a large clone that expresses two chains of the same type, it is unlikely that it is caused by technical artifacts such as multiplets, PCR chimeras, etc and it might be a biological event. On the other hand, cases where we see a more complex mixture of chain types such as 4 or more productive contigs are biologically unlikely and may be attributed to technical artifacts.

In older Cell Ranger versions (3.0 or lower) outputs, you may find these clonotypes output by Cell Ranger and can choose to ignore these clonotypes from downstream processing. Cell Ranger version 3.1 or higher assigns low confidence to all contigs in cells that express more than 4 productive chains as these are less biologically likely. The cells with 4 or more productive contigs are therefore not included in the output clonotypes.

Question:When running mkfastq I encountered the error "No bcl2fastq found on path. demux requires bcl2fastq v2.17 or greater for RTA version: 3.4.4". How can I fix this?

Answer:This indicates that mkfastq could not find bcl2fastq on your PATH environment variable. To investigate:

# Check if you have bcl2fastq and what version$bcl2fastq --versionBCL to FASTQ file converterbcl2fastq v2.19.1.403Copyright (c) 2007-2017 Illumina, Inc.

If the command above indicates that you do not have bcl2fastq installed, you will need to install it first. (See this article for help on installing bcl2fastq). After installing:

# Find where bcl2fastq is located. Below tells me the directory where bcl2fastq lives$which bcl2fastq/usr/local/bin/bcl2fastq# Check if bcl2fastq is on your PATH.# Below tells me that /usr/local/bin/ is on my PATH variable where my bcl2fastq lives$echo $PATH/mnt/home/user1/bin:/usr/local/bin:/bin:/usr/bin:/usr/local/sbin# If the path to bcl2fastq is not in PATH variable, you will need to add it.# You can add it as below.# A more appropriate way would be to add the path in your .bashrc fileexport PATH=$PATH:/use/local/bin

Question:What are the imaging system requirements for running Visium?

Answer: The imagining requirements for running Visium are as follows:

a) The three microscope systems listed below were used by 10x Genomics. Any equivalent system with the listed features may be used:

Nikon Eclipse Ti2 with brightfield and fluorescence capacity (TRITC)

Molecular Devices ImageXpress Nano Automated Cell Imaging System

b)Microscope features needed:

Objectives: 4X (Plan APO ; NA 0.20); 10X (Plan APO ; NA 0.45); 20X (Plan APO ; NA 0.75)

Scanning Stage: Microscope tile scanning functionality is required for imaging tissue sections placed on a Capture Area of a Visium Spatial slide.

Brightfield Features:

Color camera (3 x 8 bit, 2424 x 2424 pixel resolution)

White balancing functionality

Capture resolution minimum of 2.18 m/pixel

Exposure times 2 ms - 10 ms

c)Fluorescence Features (required only for Tissue Optimization workflow):

Light source (or equivalent) with a wavelength range of 380-680 nm

Monochrome camera (14 bit, 2424 x 2424 pixel resolution)

TRITC filter cube (Excitation 542/20, Emission 620/52)

Capture resolution minimum 2.18 m/pixel

Exposure times 100 ms- 2 sec

d)Additional Specifications:

Computer with sufficient power to handle large images (0.5-5 GB)

Image stitching software (microscopes software or equivalent, like Image J)

Save image in tiff (preferred) or jpeg format

For more details, please refer to our Imaging Guidelines Technical Note

Product: Visium

Question:Is a color camera required for Hematoxylin & Eosin (H&E) imaging, or can a monochrome camera be used?

Answer:We recommend using a color camera for imaging of the H&E stained tissue section as it will be difficult to see tissue morphology when using a monochrome camera. However, the Space Ranger analysis pipeline will be able to process the monochrome image.

Product: Visium

Question:How are the tissue sections fixed to the capture areas on the Visium slide?

Answer:Tissue sections are fixed to the Visium slide using cold 100% methanol.

Product: Visium

Question:Can alternative staining methods be used instead of H&E?

Answer:We have not tested alternative staining methods. Only H&E staining has been validated at this time.

Product: Visium

Question:Is Visium compatible with cells or tissues expressing GFP/RFP?

Answer:We have not tested in-house whether GFP or RFP expressing tissues are compatible with Visium. This is something we plan to test in the future.

It is unknown whether the GFP signal would be killed by the methanol fixation, but if you were to try this, the fluorescence imaging would most likely need to occur before H&E staining; especially due to the autofluorescence from the H&E.

An alternative solution to using the GFP/RFP signal directly on a Visium slide is to perform the fluorescent imaging on a subsequent section on a plain glass slide and overlay this with the H&E image generated from the Visium slide. Unfortunately, with this method you will only get a general GFP+ cell distribution, as opposed to a direct 1:1 relationship.

Product: Visium

Question:Can I use alternative methods to qPCR such as Nanodrop or Qubit for cDNA quantification in the Visium assay?

Answer:qPCR is the only validated method to accurately quantify unamplified cDNA and can't be replaced with alternative methods.

The concentration of cDNA is below the level of detection for alternative methods, therefore, qPCR has to be employed in order to quantitate how many cycles should be performed for cDNA amplification.

Product: Visium

Question:Which qPCR systems can be used for the Visium assay?

Answer: The qPCR systems that can be used for Visium include:

Applied Biosystems: QuantStudio 12K Flex system (PN-4471087)

Bio-Rad CFX96 Real-time System (PN-1855096)

Systems equivalent to those above that are compatible with 10 l reaction volumes

Product: Visium

Question: What is the structure of the final Visium library?

Answer:The structure of the final Visium library is as follows:

Product: Visium

Question:What does a good Visium Spatial Gene Expression library look like?

Answer:The trace of a good Visium library will appear as a single discrete peak approximating a bell curve 300 to 600bp in size with an average library size of approximately 450bp.You may observe 2 or 3 very small low molecular weight peaks (50 to 150bp size range) which are expected. These are low levels of primer carryover remaining after SPRIselect cleanup. This is normal and does not affect sequencing or application performance.

Example of Visium library traces from Mouse Brain tissue is provided below. Traces can also be found in the Visium Spatial Gene Expression User Guide Appendix.

Bioanalyzer - DNA High Sensitivity Kit

LabChip GX Touch HT - DNA HS Reagent Kit

Tapestation - High Sensitivity D5000 Kit

Product: Visium

Question:Can I use the Chromium i7 Multiplex Plate (PN-2201013) for Sample Index PCR with Visium?

Answer:The Chromium i7 Multiplex Plate is not compatible with Visium because it only contains a single i7 index in each well.Visium requires a dual index plate that contains a mix of one unique i7 and one unique i5 sample index per well.

You will need to purchase the Dual Index Kit Plate TTSet A, 96 rxns (PN-1000215)

Product: Visium

Question:What index plate do I need for performing Sample Index PCR with Visium?

Answer:Visium requires Dual Index Kit Plate TT Set A (PN-1000215).Visium requires a dual index plate that containsa mix of one unique i7 and one unique i5 sample index per well.

Product: Visium

Question:Can Visium libraries be pooled with other 10x libraries?

Answer:Visium libraries are dual-indexed and therefore should not be pooled with other 10x single indexed libraries.

Product: Visium

Question:What are the minimum recommended sequencing specifications for Visium libraries?

Answer: The recommended sequencing depth for the Visium library depends on several factors (tissue complexity, tissue coverage, experimental goals) and can be calculated as per the example below.

Werecommend a minimum of 50k read pairs per spot covered with tissue.

Estimate the % of capture area covered by the tissue section based upon the H&E brightfield image.

(% tissue coverage area * 5000 total spots in the capture area) * 50,000 reads pairs per spot

Example: 50% of capture area covered by tissue section

(.50 tissue coverage * 5000 total spots) * 50,000 read pairs per spot

= 125 million read pairs (250M total reads)

Product: Visium

Question:What is OCT?

Answer:Optimal Cutting Temperature compound (OCT) preserves the structure of the tissue and provides support during cryosectioning. OCT is water-soluble and does not penetrate the tissue. It is washed away during the Hematoxylin and Eosin staining.

Product: Visium

Question:Are tissues flash-frozen in liquid nitrogen compatible with Visium?

Answer:Tissues flash-frozen in liquid nitrogen experience uneven freezing which can result in tissue cracking. We recommend flash-freezing tissues using an isopentane and liquid nitrogen bath. Refer to our Tissue Preparation Guide for additional details.

Product: Visium

Question:How should I prepare fresh tissue for shipment for making a Visium slide?

Answer: In order to ship fresh tissue for use in making a Visum slide, the following guidelines are recommended:

Snap freeze the fresh tissue in an isopentane and liquid nitrogen bath

Place the frozen tissue in a pre-cooled sealed container sitting on dry ice using pre-chilled forceps or spatula

Store the sealed container at -80C until shipment

Samples can be shipped overnight in dry ice, provided there is enough dry ice to account for transit and delivery times.Refer to the local institution or delivery service for detailed instructions on shipping samples in dry ice.

Once the package has been received, the sealed container should be transferred on dry ice to a -80C freezer for storage.

For detailed instructions on how to snap freeze tissue, please refer to our Tissue Preparation Guide.

Product: Visium

Question: What tissue types have been tested using Visium Slides?

Answer:Visium utilizes unbiased polydT capture and therefore should be compatible with most mRNA containing tissues.We have tested a variety of human, mouse. and rat tissues. Please see the following link for the most up-to-date list:

Visium Spatial Gene Expression Optimized Tissues

For tissues that are not listed, we recommend optimizing these tissues using the Visium Spatial Tissue Optimization kit before proceeding with the Visium Spatial Gene Expression kit.

Product: Visium

Question:Can I run multiple tissue sections in one capture area of the Visium Slide?

Answer:We currently support one tissue section per capture area on the Visium Slide.

Product: Visium

Question:What tissue section thickness should I use with the Visium Slides?

Answer: The thickness of sectioned samples for the Visium Slidewill depend upon tissue type. Fatty tissues generally require thicker sections.5 - 35 um sections have been tested in-house, however, 10 um sections are used for most tissue types.Changes in tissue section thickness will require a reassessment of permeabilization time using the Tissue Optimization Kit.

Product: Visium

Question: Areformalin-fixed paraffin-embedded (FFPE) tissues compatible with Visium?

Answer:Only fresh frozen tissues embedded in OCT have been validated with Visium.We are currently working on an FFPE solution.

Product: Visium

Question:Is Visium compatible with immunohistochemistry (IHC)?

Answer:Visium is currently only compatible with Hematoxylin and Eosin staining; however, weare working on developing IHC compatibility.

Product: Visium

Question:What is the expected Visium Spatial Gene Expression Slide spot dropout rate?

Answer: Spots on the Visium Spatial Gene Expression Slide are printed with >99% accuracy.

Product: Visium

Question:How do I assemble the Visium slide into the Slide Cassette?

Answer:Assembly of the Visium slide into the Slide Cassette can be done using the Slide Alignment Tool found in the Visium Accessory Kit, or performed manually; whichever method is easiest for assembling the Visium Slide inside the the Slide Cassette.Instructions for both methods are included in both the Visium Spatial Tissue Optimization and Gene Expression User Guides.How-to-Videos demonstrating how to place the slide in the Slide Cassette with the aid of the Slide Alignment tool and without are also available. Please see below GIF demonstrations.

Manual Assembly

Assembly Using Slide Alignment Tool

Product: Visium

Question:Does 10x recommend a model system to useD as a control for practicing on prior to using a real sample with Visium?

Answer:Mouse brain is a good model tissue for the Visium assay if you want to get acquainted with sample preparation and/or the Visium workflow. Mouse brain is commercially available from different vendors depending on where in the world you are located.

In addition, control RNA should be used as cDNA synthesis positive control when performing the Tissue Optimization Kit

Product: Visium

Question:Can I modify the Visium Spatial Gene Expression solution to be targeted?

Answer:Targeted or custom Visium assays are currently not supported by 10x.We are working on a targeted solution.

Product: Visium

Question:Which thermal cyclers is the Thermocycler Adapter compatible with?

Answer:

The Thermocycler Adapter is compatible with the following instruments:

Bio-rad C1000

BioRad S1000

Bio-rad T100

Eppendorf Mastercycler EP Gradient S

Thermo Fisher Scientific Veriti

Eppendorf Vapo Protect

MJ Tetrad 2

The Thermocycler Adapter is not compatible with the following instruments due to the lid not being able to close and a heated lid is required to run the Visium workflows.

Applied Biosystems ProFlex

Applied Biosystems SimpliAmp

Product: Visium

Question:What is included in the Visium Accessory Kit?

Answer:The Visium Accessory kit is required to run the Visium Spatial Tissue Optimization and Gene Expression workflows and is generally a one-time purchase.The kit includes the following 4 items:

1) Thermocycler adapter that up to two slide cassettes fit onto for direct heat transfer from the thermal cycler block to the Visium slides

2) Imaging Test Slide for the user to determine if their imaging system is compatible with the Visium solution. Once system compatibility has been determined, the slide should be used to set-up programs for imaging of the Tissue Optimization and Gene Expression slides. Refer to our Imaging Guidelines for additional instructions.

3) Slide Alignment Tool is included which aids in placing the Visium slide into the disposable Slide Cassette

4) 10x Magnetic Separator for performing SPRI bead purifications when processing the Gene Expression slide

Product: Visium

Question:Do the spatial barcodes differ across individual capture areas on the Visium Spatial Gene Expression Slide?

Answer:The 4992 spatial barcodes are the same in each of the 4 capture areas on the Visium slide, therefore customers will need to generate a separate dual-indexed library for each of the capture areas.

Product: Visium