10x Genomics Single-Cell Processing
The 10x Genomics Chromium Controller is a single-cell profiling technology that enables the analysis of large cell numbers at a high capture efficiency (of up to 65%). The platform allows for high-throughput analysis in a variety of cell types as well as single-cell nuclei. The workflow encapsulates cells or nuclei together with gel beads into nanodroplets (single-Poisson distribution loading). Trained core staff will prepare the libraries from single-cell suspensions submitted by the research investigator.
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Flavors of 10x Genomics Single Cell Assays
| Assay | Gene Expression | Immune Profiling | RNA Flex | ATAC | Multiome (Gene Expression + ATAC) |
|---|---|---|---|---|---|
| Description | 3' or 5' gene expression | 5’ gene expression with V(D)J | probe-based whole transcriptome gene expression | chromatin accessibility | simultaneous detection of mRNA and chromatin accessibility |
| Sample Compatibility | cells or nuclei | cells | fixed cells, fixed nuclei, fixed tissue, FFPE tissue | nuclei | nuclei |
| Additional Multiomic Capabilities | Cell Surface Protein CRISPR Screening | Cell Surface Protein Antigen Specificity BCR/TCR Sequencing CRISPR Screening | Cell Surface Protein | none | none |
| Plexing Compatibility | yes | yes | yes | no | no |
| Cell Throughput | Standard: 10,000 cells per channel; up to 80,000 cells per chip; 140,000 cells per chip with plexing High: 20,000 cells per channel; up to 320,000 cells per chip; up to 730,000 cells per chip with plexing | Standard: 10,000 cells per channel; up to 80,000 cells per chip; 140,000 cells per chip with plexing High: 20,000 cells per channel; up to 320,000 cells per chip; up to 730,000 cells per chip with plexing | Singleplex: 10,000 cells per channel; up to 80,000 cells per chip Multiplex: 128,000 cells per channel; up to 1,024,000 cells per chip | Standard: 10,000 cells per channel; up to 80,000 cells per chip | Standard: 10,000 cells per channel; up to 80,000 cells per chip |
| Species Compatibility | diverse | human or mouse | human only | diverse | diverse |
| Optimal Concentration Ranges | Standard: 700-1200 cells/ul High: 1300-1800 cells/ul | Standard: 700-1200 cells/ul High: 1300-1800 cells/ul | NA - minimum total of 50K cells | 3000-8000 nuclei/ul | 3000-8000 nuclei/ul |
| Buffer Requirements | PBS+0.4% BSA recommended Other standard media with no more than 10% FBS or 2% BSA; No EDTA | PBS+0.4% BSA recommended Other standard media with no more than 10% FBS or 2% BSA; No EDTA | Quenching Buffer + Enhancer + 50% Glycerol from 10X Fixation kit | 1X Nuclei Buffer | 1X Nuclei Buffer with RNase Inhibitor and DTT |
Single-cell data are analyzed with the Cell Ranger and Loupe Cell Browser software.
Sample Submission Instructions
Feature Barcoding
Feature Barcoding is the broad term used by 10x Genomics in order to describe any method that adds extra layers of information to cells by running single cell gene expression in parallel with other assays to gain useful biological information. Mostly this comes in two forms – looking for cells that are expressing certain proteins on the cell surface (CITE-seq) or using ubiquitous markers in order to pool different samples together into one channel of the chip (Cell Multiplexing/Cell Hashing).
Cell Multiplexing
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Cell or nuclei samples can be labeled with a molecular tag and subsequently mixed with other labeled samples. The set of multiplexed samples are processed together in a single GEM well. After cell encapsulation, library preparation, and sequencing, molecular tag information is assigned to cells. Tag assignment enables identification of droplets that originally contained one (singlet) or more cells (multiplets). Cells assigned a given single tag are binned together, bioinformatically recapitulating the individual samples originally mixed together.
Advantages of Cell Multiplexing include:
- Increased sample throughput in a single experiment
- Increased number of cells assayed in a single experiment
- Increased number of possible replicates in a single experiment
- Detection of multiplets and their removal prior to analysis
Surface Protein Expression
As part of their TotalSeq antibody catalog, BioLegend offers this technology through antibody-oligonucleotide conjugates that can be used for both CITE-seq and Cell Hashing. Antibodies come in three formats that differ in their capture sequence for library preparation so pay close attention to which antibody catalog you use. These are limited to Human and Mouse samples.
- TotalSeq A is compatible with any Single Cell RNA-seq product that uses poly(dT) for mRNA capture. This is the format originally used in the CITE-seq protocol. At this time, the 10X Genomics analysis pipeline (CellRanger) does not support the processing of the protein derived libraries. Data must be analyzed using the R based Searat package.
- TotalSeq B is specifically designed to be compatible with the newer 10X Genomics Single Cell 3’ v3 reagents used in the core and is fully supported by the 10x Genomics CellRanger software. These antibody oligos are complementary to a unique capture sequence on the Single Cell 3′ v3 Gel Beads so antibody barcodes do not compete for the oligo(dT)s capturing the mRNA.
- TotalSeq C is specifically designed to be compatible with the 10X Single Cell 5′ workflow used in the Immune Profiling kit. It is also fully supported by 10x Genomics CellRanger software run.
If using 10X Genomics single cell 3′ GEX, TotalSeq B antibodies and/or hashtags are the best way to go!
Frequently Asked Questions
How many cells should I target for capture and how many reads/cells do I need?
This 10X Genomics Technical Note discusses how sequencing depth and cell number influence the detection of major cell types in peripheral blood mononuclear cells (PBMCs).
Can I have my cells checked without running my full experiment?
Yes, we offer viability only appointment slots for you to check your cell dissociation method before using experimental cells. Select “viability/count only” as your assay type when filling out the MiCores 10X appointment request form. We ask that you bring a maximum of four samples for the appointment.
How much sequencing do my samples need?
The 10X minimum recommendations are as follows:
- ATAC – 25,000 reads/cell
- Gene Expression (3’ and 5’) – 20,000 reads/cell
- Immune Profiles (B and T cells) – 5,000 reads/cell
- ADTs and HTOs – 5,000 reads/cell
What sort of buffers can my samples be in?
This is dependent on which analysis you would like to do.
- ATAC – Your nuclei must be in 1X Nuclei Buffer from 10X Genomics. We have this buffer available for you to pick up from us prior to your appointment time.
- CNV (Single Cell DNA/Copy Number Variation) – Cells must be in a buffer that contains no more than 0.04% BSA or 10% FBS. Supported buffers and media include: PBS+0.04%BSA, HBSS+0.04%BSA, EMEM+10%FBS, DMEM+10%FBS, RPMI+10%FBS, Hams F12+10%FBS, IMDM+10%FBS, DMEM:F12+10%FBS
- Gene Expression and Immune Profiling – Both of these assays are compatible with a wide variety of buffers and media. Some examples are: EMEM+10%FBS, DMEM+10%FBS, RPMI+10%FBS, Hams F12+10%FBS, DMEM:F12+10%FBS, M199.
Can I FACS sort my cells before I bring them?
Absolutely. Some things to keep in mind when sorting your cells prior to bringing them:
- FACS will overestimate the number of cells on average by 50%. One way to mitigate this problem is to stain your cells with Ruby. This will separate the cells from debris and make the count much more accurate.
- Your cells may not like to stay in FACS buffer. You can sort into a full media that is compatible with your cells, and resuspend them in a compatible media afterward.
- Your cells will likely be very dilute when they come out of the sorter. You will likely want to concentrate them before coming to the Core.
Can we do CITE-seq or cell hashing?
Yes, we support CITE-seq (ADT) and cell hashing (HTO) libraries. 10X Genomics refers to these as Feature Barcoded libraries. We primarily work with the BioLegend TotalSeq suite of antibodies, but are willing to work with your project. One recommendation is that if you are FACS sorting, simultaneously stain your cells with the FACS antibodies as well as the single cell antibodies.