Single Cell Expression Profiling & Genomics (10X Genomics, Fluidigm)

The 10X Genomics Single Cell system is the single-cell expression profiling platform enabling the analysis of large cell numbers at the highest capture efficiency (of up to 65%). The technology allows for high-throughput single cell transcriptomics of many different cell types as well as single-nuclei expression profiling. The flexible workflow encapsulates 100 to 15,000 cells or nuclei per library together with micro-beads into nano-droplets.  Each bead is loaded with adapters containing one of  750,000 different barcodes for the single cell RNA-seq library preps.  In contrast to other protocols (e.g. Drop-Seq) the 10X controller is capable of loading “all” droplets with micro-beads, enabling single-Poisson distribution loading and thus high capture efficiencies (in contrast to double-Poisson loading of other protocols). The single-cell encapsulating process is significantly faster compared to inDrop or Drop-Seq. Up to eight samples can be processed per batch within minutes. The resulting data can be analyzed with the free Cell Ranger and Loupe Cell Browser software. In addition the Bioinformatics Core has developed a custom single-cell data analysis pipeline for 10X data.

  • By default, our staff will prepare the single-cell RNA-seq (scRNA-seq) libraries from cell suspension samples submitted by the research labs.  Since the samples should be processed as quickly as possible, the experiments need to be planned and scheduled together with our staff.
  • In case your lab is planning a large number of single-cell experiments, it might make sense for investigators to get trained to be able to process the cell suspensions on the 10X Genomics Chromium controller.

Tsne-plot_single-cell

The principles of the 10X Single-Cell RNA-seq library preparation:

10XGenomicsSingleCell\

10X Chromium Single Cell Features:

  • Fast workflow from cell suspension to 3′-cDNA library.
  • Captures 100-80,000+ cells (from up to 8 samples) in < 7 minutes.
  • Recovers up to  ~65% of cells (typically 50%).
  • Low doublet rate (~0.9% per 1,000 cells).
  • Compatible with Illumina HiSeq4000, NextSeq, and MiSeq sequencers.
    The 10X Single-Cell libraries are most  economically sequenced on the Illumina HiSeq 4000 with paired-end 100bp reads or on the NextSeq with the 150 cycle kits.
  • For the most applications an average of 50,000 reads per cell should be sequenced (for cell types with complex transcriptomes). For isolated nuclei 25,000 reads each are recommended,
  • It is possible to work with cryo-preserved cells, enabling safe sample shipping and batching.
  • In addition to cell suspension samples also nuclei suspensions can be studied, enabling the analyses of brain tissues.

10XChromium

For information regarding sample preparation, please refer to the 10X cell preparation guide.
Please inquire with our 10X Genomics specialist Diana Burkart-Waco, PhD, for further details.

Our Countess II Automated Cell Counter (Life Technologies) can assist you with the preparation and QC of the cell suspension. It uses trypan blue staining to provide total cell counts, reports viability, and measures average cell size in as little as 10 seconds.

CountessII-cell counter499x180

 

Both Single-Cell-Genomics and Single-Cell-Transcriptomics are enabled by  the Fluidigm C1 Single Cell Automation Prep. This is a single cell capture microfluidic technology that processes up to 500 single cells to extract single cell transcriptome, reverse transcribe, pre-amplify and ultimately detect and analyze cell activity at the single cell level.  Protocols for genomic analysis of single cells are under development.

C1plate                                            c1_left_250x225