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Single-Cell Expression Analysis (scRNA-Seq)

10X Genomics Chromium Single-Cell System

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 20,000 cells or nuclei per library together with micro-beads into nano-droplets.  Each bead is loaded with adapters containing one of  750,000 different 10x Genomics GemCodes (i.e., 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.

  • Full-Service Workflow: 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 well in advance with our staff. 
  • Self-Service Workflow: 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 it is difficult to plan well in advance when your cells would be ready.

The workflow of the 10X Single-Cell RNA-seq library prep:

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 < 20 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 the Illumina NovaSeq 6000 with paired-end 100 bp reads with the 200 cycle kits. (The assay requires at least a 28 cycle forward read (10X GEMcode & UMI, an 8 bp read (i7 index), and a 91 cycle reverse read (insert).
  • For most expression applications, an average of 20,000 reads per cell or nuclei should be sequenced (for cell types with complex transcriptomes). 
  • 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 suspension scan also be used. For information regarding sample preparation, please refer to the 10X cell preparation guide.

Full-Service Workflow: Cell Sample Submission Requirements 

  • Ideally, plan to provide at least 20 ul of at a concentration of 1000 cells/ul to target 5000-10,000 cells for loading from submitted cell suspensions. The cells should be suspended in PBS with 0.04% BSA or an acceptable alternative buffer or media.
  • Users should count their cells before they come to the lab.  There is a Luna FL automated cell counter available at the Genomic Division laboratory if needed.
  • Cells should be dissociated, debris free, and greater than 90% viability-but viability should not fall below 70%.
  • The most important factors are quantifying accurately the cell concentration and determining the cell viability.
  • We acknowledge and realize that not everyone can achieve that concentration - especially if obtaining cells via flow cytometry or some sort of cell sorting procedure.  
  • Bring your cell suspension samples by or before 2 PM to start the procedure on the day your project is scheduled.

Self-Service Workflow: Cell Sample Submission Requirements

  • Investigator will contact the Genomics Division to reserve time on the instrument
    • Let us know if after hour access is needed 
    • User will be need to complete training on the 10X website 
    • Once training is complete, meet with the Genomics Division to discuss access and go over the use of the instrument
  • Investigators will be responsible for purchasing the 10X reagents, preparing their cells, operating the 10X chromium system and preparing their libraries
  • The Genomics Division will provide support in the various bioanalyzer quality assessments and answer questions.  Division personnel will not be performing the library preparation.
  • What is provided: 10X Genomics Controller, Cartridge holder, Pipets and tips, Strip tubes, SPRI beads-enough for 8 reactions, 50% Glycerol, Microfuge, and Vortexer.  Luna FL automated cell counter and thermocycler (adjustable temperature lid and accommodates a 100 ul rxn volume) are available, if needed.
  • What is NOT provided:  10X Reagents (e.g., beads, cartridges, index plate and primers), EB buffer, LoTE, Tween-20, Cold Block for strip tubes, and magnet (Product #: 120250 10x Genomics) to capture beads.

Sample Submission: All projects must be scheduled in advance.  First-time users should contact Kevin Knudtson to discuss experimental design, sample submission and workflow. Contact the Genomics Division at 335-7928 to schedule your project.

Please complete the File10X Sample Submission Form and bring with your samples.

 

Fees

Full-Service Workflow Fees

Library Type Sample No. Campus Cost/Sample Off-Campus Cost/Sample
3' Expression-Single-Cell RNA-Seq using 10X Chromium System (v3.1) 1 $2,492 $2,769
  2-8 $2,235 $2,512
5' Expression-Single-Cell RNA-Seq using 10X Chromium System (2) 1 $2,557 $2,843
  2-8 $2,300 $2,577
5' Expression-Single-Cell RNA-Seq + V(D)J using 10X Chromium System (2) 1 $3,022 $3,425
  2-8 $2,765 $3,168
V(D)J enrichment (only) using 10X Chromium System (v2) 1 $2,753 $3,072
  2-8 $2,496 $2,815
10X ATAC-Seq (v1.1) 1 $2,245 $2,480
  2-8 $1,988 $2,223
10X Multiome Expression and ATAC-Seq 1 $3,793 $4,154
  2-8 $3,535 $3,896
10X Genomics Library-cDNA only, Failed Yield 1 $2,187 $2,338
  2-8 $1,930 $2,081

Self-Serve Workflow Fees

  • Access to 10X controller and cartridge Holder: $200/run (10X consumables not provided)
  • Bioanalyis: Full Chip URGENT $93.00 (pricing for cDNA TAT) and $10.00 per sample (pricing for final libraries)

 

Support Documents

 

10X Single-Cell Preparation Guide

Technical Note: Guidelines or Accurate Target Cell Counts

Alternative Buffer and Media Cell Suspension Options