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Single-Cell Profiling (RNA-Seq and ATAC-Seq)

10X Genomics Chromium Single-Cell System

#Fee Schedule

#Resources

 

The 10X Genomics Chromium Single-Cell System is used to provide a single-cell expression profiling technology that 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 10,000 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.  Up to eight samples can be processed per batch. The resulting data can be analyzed with the free Cell Ranger and Loupe Cell Browser software. In addition the IIHG Bioinformatics Division has developed a custom single-cell data analysis pipeline for 10X data. 

The service operates the 10X Genomics Chromium X and iX Controllers. These controllers support Single-Cell Gene Expression, Immune Profiling,  ATAC, and other assays that are supported by the Chromium X controllers. These assays include the measurement of gene expression, cell surface proteins, immune clonotype, antigen specificity, and/or chromatin accessibility.

There are two service workflows available:

Full-Service:

Our staff will prepare the single-cell (or nuclei) RNA-seq libraries from cell/nuclei suspension samples submitted by the research labs.  Since the samples should be processed as quickly as possible, these experiments need to be planned and scheduled well in advance with our staff. 

Self-Service:

The investigator performs all the necessary steps to prepare their single-cell libraries for sequencing.  This option may be beneficial if your lab is planning a large number of single-cell experiments or if it is difficult to plan well in advance when your cells would be ready.

Full-Service Workflow

Step 1 - Scheduling Email garry-hauser@uiowa.edu and mary-boes@uiowa.edu
Step 2 - Prepare Samples

Suspend cells in PBS with 0.04% non-acetylated BSA or other 10x approved buffer/media

Cell viability should be >90% (minimum 70%)

Contact us at least 30 minutes before you bring your cells/nuclei
Step 3 - Fill out the form Complete the 10x Single Cell Full Service Workflow form - File Single Cell Full Service Submission Form.xlsx
Step 4 - Drop off your samples

Bring the completed and printed sample submission form

Arrive with your samples before 2:00pm on the day you are scheduled.

Self-Service:

Training Schedule Get ready We will You will

Complete video training on the 10x website: https://www.10xgenomics.com/videos

Contact us to reserve the instrument at least two business days in advance

Email:

mary-boes@uiowa.edu and garry-hauser@uiowa.edu

You must provide

  • 10x reagents
  • EB buffer
  • LoTE
  • Tween-20
  • Cold blood for strip tubes
  • Magnet to capture beads
  • Answer questions
  • Provide support with bioanalyzer QA
  • Provide access to
    • 10x Genomics Controller and Cartridge holder ($245 fee)
    • Pipettes and tips
    • Strip tubes
    • SPRI beads
    • 50% glycerol
    • Microfuge and vortexer
    • By request: Luna FL automated cell counter and thermocycler (100ul rxn volume)
  • Arrive at your scheduled day and time
  • Bring the required materials
  • Prepare and count the cells
  • Operate the 10x Chromium controller
  • Prepare libraries for sequencing
  • Prepare the NGS Sample submission form prior to submitting samples for sequencing

                                                 

 

 

 

 

Fee Schedule (effective Feb 1, 2024)

       

Resources

10X Single-Cell RNA-seq library prep workflow

10X Chromium Single Cell Features:

  • Fast workflow from cell suspension to 3′ or 5’-cDNA libraries, and T or B cell V(D)J analyses.
  • 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 NovaSeq 6000 and MiSeq sequencers.
  • The 10X Single-Cell libraries are most economically sequenced on our Illumina NovaSeq 6000.
  • At least 20,000 reads per cell (or nuclei) should be targeted for sequencing.
  • It is possible to work with cryo-preserved cells, enabling safe sample shipping and batching.
  • The cell size limit is comparatively high.  Cells can have a diameter of up to 50 µm.
  • In addition to cell suspension samples, nuclei suspensions can also be used.  For information regarding sample preparation, please refer to the 10X cell preparation guide.

Genomics Division