The 10x Genomics Chromium platform combines microfluidics with molecular barcoding to provide mRNA quantification from thousands of individual single cells. The Single Cell Gene Expression Assay is able to generate single cell transcriptomic measurements from up to 10,000 cells per cell suspension with up to 65% capture rate efficiency enabling the identification of rare cell types from heterogeneous populations. User provided single cell or single nuclei suspensions are loaded onto the Chromium iX to generate single cell barcoded gel-bead emulsions from which Illumina-compatible library preps are generated and sequenced on the NovaSeq in collaboration with UWBC DNA Sequencing Facility followed by optional analysis using a custom developed single cell data analysis pipeline by the UWBC Bioinformatics Resource Center. The resulting data can then by analyzed and explored using the freely downloadable Loupe Cell Browser software (link for Loupe Cell Browser free download, tutorial, and sample data sets).
Additionally, 10x Genomics offers assays for single cell immune profiling (sc-V(D)J enrichment kit), single cell copy number variation profiling, single cell chromatin accessibility analysis (sc-ATAC-Seq), de novo assembly, and genome or exome sequencing.
Please e-mail email@example.com to schedule a meeting to discuss your proposed experiment and timeline!
10x Genomics Single Cell Features
- Workflow from single cell suspensions.
- 8-channel microfluidic chip allowing up to 8 samples to be processed simultaneously. Samples are barcoded individually for a multiplexed sequencing reaction.
- 10x Chromium instrument capable of capturing 500 to 10,000 cells per sample; up to 80,000 cells per chip.
- Captures approximately 60% of loaded cells
- Low doublet rate: ~0.8% per 1,000 cells; ~1.6% per 2,000 cells; ~2.3% per 3,000 cells; ~3.1% per 4,000 cells; ~3.9% per 5,000 cells; etc.
- 50,000 reads per cell should be sequenced for saturating gene detection. 70,000 reads per cell is appropriate for expression analysis.
- Nuclei suspension can also be prepared and sequenced.
A note about these kits: We only maintain a stock of the 3′ Gene Expression kits. For any other assay, we will order the kits specifically for your project. We ask that you provide a funding string at the time we place the order so that we can eventually bill for the cost of the kits even if your project plans do not end up proceeding. While 10X Genomics is usually able to deliver kits a day or two after they are ordered, they do suffer from occasional back-order issues. If this happens, we will keep in touch with you to let you know when we receive the kits, so that you are not bringing over samples on a day when we do not have the reagents to process them.
Single cell/single nucleus 3′ Gene Expression – The standard “workhorse” kit for single cell/nucleus RNA sequencing. The kit employs polyA-based capture of mRNA at the 3′ end to generate dual indexed libraries containing both a cell barcode identifying the cell of origin as well as a unique molecular identifier (UMI), which will be unique to every transcript captured. Add-on modules are available for cell surface protein expression (analogous to CITE-seq), CRISPR/sgRNA screening, and sample multiplexing.
Single cell 5′ Gene Expression/Immune Profiling – This kit also generates single cell RNA-seq libraries through capture at the 5′ end by capturing the TSO sequence added to this end of the transcripts in a template-switching reverse transcription reaction. The main reason to choose this kit over the 3′ Gene Expression kit is the immune repertoire profiling add-on module, which allows for the parallel PCR enrichment and library preparation of B cell/T cell receptor V(D)J sequences. Currently, 10X Genomics only sells these modules for human and mouse V(D)J amplification. Like the 3′ Gene Expression kit, additional add-on modules are available for cell surface protein expression and sample multiplexing. A CRISPR/sgRNA screening module compatible with the 5′ Gene Expression kit is coming soon.
Single nucleus ATAC Sequencing – This kit is used to generate ATAC-seq libraries for measurement of chromatin accessibility at single-nucleus resolution.
Single nucleus Multiome ATAC + Gene Expression – This kit uses gel beads with capture oligos for both mRNA polyA tails and transposed DNA for the parallel preparation of ATAC-seq and 3′ Gene Expression libraries from the same nucleus. At approximately the mid-point of the library preparation process, the samples undergo an initial PCR amplification to produce sufficient material for library construction, and the pool is then split to produce ATAC-seq and gene expression libraries from the same initial sample. Note that due to differences in the required sequencing parameters, ATAC-seq and gene expression libraries cannot currently be sequenced on the same flow cell; as a result, the sequencing costs will be approximately double what they would be for the standalone ATAC or gene expression assays.
Visium Spatial Transcriptomics (Fresh frozen) – In this workflow, OCT-embedded tissues are cryosectioned and transferred on to a slide containing four capture areas. Within each capture area are approximately 5,000 spatially-barcoded spots. Over the course of the experiment, the tissue placed above these spots will be permeabilized, and polyA transcripts will be bound by the capture oligos within the spots, allowing users to map the location of transcripts within an image of the H&E-stained tissue. Each spot is approximately 55 microns in diameter and therefore this assay is not considered to have single cell resolution, although the forthcoming updated version “Visium HD” will reportedly feature this. Visium projects are handled in collaboration with the UW-Madison TRIP Lab, which handles the parts of the library preparation involving the tissue sections.
Visium Spatial Transcriptomics (FFPE) – Due to the likelihood of RNA degradation in FFPE-preserved tissues, this version of Visium uses a dual probe-based method of capturing transcripts and is currently only available for human and mouse. When both probes are able to bind to their adjacent target sites, they are ligated together, and this ligated probe construct becomes the template for capture on the Visium slide and subsequent library preparation. Before starting the Visium FFPE workflow, an RNA quality assessment is performed to determine the fraction of RNA in the sample that is above 200 bp in length (“DV200”); for best results, this value should be at least 50%. This assessment can also be done by the GEC for users who lack access to an instrument such as the Bioanalyzer or Tapestation. As with fresh frozen Visium, the work with the tissue sections is done by the TRIP Lab.
Information on the probe sets and the number of targeted genes can be found here.
(The images in this section were taken from their respective User Guides provided on the 10X Genomics website.)
For more detail, please see the GEC’s Guide to Getting Started with 10X.
Single Cell Suspension Submission Guidelines
- Bring cells over on ice along with extra cell suspension buffer.
- Minimum concentration is 300 cells/ul (700-1200 cells/ul optimal) with at least 50,000 cells total in limited cell source situations. Projects not limited in source material should submit as many cells as possible to avoid loading shortages (at least 100,000 if not sorted or 150,000 – 200,000 if sorted).
- Required volume depends on cell concentration and targeted cell recovery amount plus an additional 10ul for cell counting. See the Cell Suspension Volume Table for details regarding input cell suspension volume.
- Recommended cell suspension buffer is 1x PBS (Ca++/Mg++-free) + w/v 0.04% BSA (non-acetylated) + (up to) 1U/ul RNase Inhibitor (optional for cells; required for nuclei. Sigma Aldrich PN-3335399001 recommended). Up to 2% BSA can be used if cellular aggregation is a concern. Additional buffers are also compatible with the assay (section 1.6 of Cell Prep Guide). Cell suspension buffers should also be free of EDTA, DNase/RNase, and surfactants.
- We advise that single cell suspension should be at least 75% viable, and free of visible debris and multiplets.
- Cell concentration, viability, and singlet frequency is measured using the Luna-FX7 Automated Cell Counter, which can measure viability by both trypan blue exclusion and AOPI fluorescence. We are happy to schedule “mock measurements” using the Luna to evaluate test samples prior to your real submission to ensure that maximum sample prep quality is achieved.
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All data and services provided by the University of Wisconsin Biotechnology Center Gene Expression Center are intended for research purposes only. Services are not intended nor certified for diagnostic or clinical use.