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University of Wisconsin–Madison

Gene Expression Center

10X Single Cell RNA Seq Services

10x picture

The 10x Genomics Chromium Single Cell Gene Expression Assay combines microfluidics with molecular barcoding to provide digital 3’ 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 Controller 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 (see support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/tutorial 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.

Documents for download: scRNA-Seq Fillable Submission Form_v5

Please e-mail gecinfo@biotech.wisc.edu to schedule a meeting to discuss your proposed experiment and timeline!

Library Preparation
Loupe

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 100 to 10,000 cells per sample; up to 80,000 cells per chip in ~7 minutes.
  • Recovers up to 65% of cells (typically closer to 50% depending on cell type)
  • 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.

Single Cell Suspension Submission Guidelines 

  • Bring cells over on ice along with extra cell suspension buffer.
  • Minimum concentration is 100 cells/ul (700-1200 cells/ul optimal).
  • 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) + 1U/ml RNase Inhibitor.  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 and DNase/RNase.
  • We advise that single cell suspension should be at least 70% viable (trypan blue exclusion) (exceptions apply), and free of visible debris and multiplets.
  • Cell concentration, viability, and singlet frequency is measured using the Countess II Automated Cell Counter.  Training is available is to use the cell counter prior to performing a scRNA-Seq experiment to ensure that maximum sample prep quality is achieved.
Cell Count
To request a quote please contact sandrasb@biotech.wisc.edu
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.