Currently the core provides services on two single cell platforms: the 10X Chromium system and pooled scRNA-Seq in 96-well plates. The two methods accommodate different levels of throughput, providing flexible options to U-M investigators.
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 per sample together with gel beads into nanodroplets (single-Poisson distribution loading). For most assays, up to eight samples can be processed per run allowing for capture of up to 80,000 cells per run. Trained core staff will prepare the libraries from single-cell suspensions submitted by the research investigator. Cells can be suspended in a wide range of media or buffers. Please see the 10X Sample Prep Guidelines for recommendations.
- Cell Viability needs to be >90%
- Cell concentration needs to be 750-1200 cells/μl and should be determined via an automated counter. If you have questions regarding cell sorting, counting, viability, please talk to the Flow Cytometry Core.
The resulting data are analyzed with the free Cell Ranger and Loupe Cell Browser software. The full list of 10X solutions are listed below:
Single Cell Gene Expression (SC3′)
Single cell expression measurements that enable discovery of gene expression dynamics and molecular profiling of individual cell types. https://www.10xgenomics.com/solutions/single-cell/
Single Cell Immune Profiling (VDJ)
Profiling full-length paired V(D)J transcripts from lymphocytes on a cell-by-cell basis. Can be coupled with 5’ gene expression measurements to provide high resolution insights into the adaptive immune system. https://www.10xgenomics.com/solutions/vdj/
Single Cell Copy Number Variation (CNV)
Reveals genome heterogeneity and clonal evolution. Study disease pathogenesis or characterize neuronal mosaicism at the single cell level. https://www.10xgenomics.com/solutions/single-cell-cnv/
Single Cell Assay for Transposase Accessible Chromatin (ATAC)
Genome-wide chromatin accessibility information for tens of thousands of nuclei. Elucidate gene regulatory mechanisms to understand epigenetic heterogeneity at high resolution. https://www.10xgenomics.com/solutions/single-cell-atac/
Direct Cell Lysis
For full transcriptome single-cell sequencing, users can work with flow-cytometry to sort individual cells into individual wells containing lysis buffer on a 96-well plate. After sorting, the plates can be spun down and frozen until such time that the user is ready to submit samples to the core for library prep and sequencing. Library prep uses a template switching method to generate full length cDNAs and subsequent library construction. This process provides full length transcript coverage, is compatible with standard RNA-Seq pipelines, and allows for splicing and SNP detection.