Skip to main content

10x Single Cell 3' RNA Sample Submission Requirements:

Our facility offers a full range of services for library construction and sequencing with the NovaSeq 6000, MiSeq, Oxford Nanopore GridION and the 10x Genomics Chromium. The information below is for 10x Genomics Single Cell work only.  Please return to our main Sample Submission page for all other sample types.

Contact Information:

  • All inquiries about 10x Single Cell should be directed to ngsequencing@illinois.edu.  This email will copy both Chris Wright and Alvaro Hernandez so you receive the quickest reply possible.

  • Timing is critical. Please contact the DNA Services lab BEFORE you begin growing your cells or your experimental study animals to schedule test counts and setup dates. This will ensure the lab will have staff available to process your samples.  Currently, this protocol is in high demand. Scheduling usually occurs:

    • 10x single cell test counts: ~1-2 weeks from when you contact us

    • 10x library construction: ~4-8 weeks from when you contact us 

  • Below, you'll find a host of details and links that you can refer to throughout this process.

Basic Information:

  • 10x Genomics Single Cell libraries can provide incredibly powerful data by allowing interrogation of highly heterogeneous tissues on an individual cell level.  Click the 10x Genomics link to read more about the technology and  it's various applications.

  • The general process is as follows:

    1. Determine experimental needs --> meet with us, and with HPCBio if you need analysis help.

    2. Test tissue dissociation protocols.

    3. Set up counts with DNA Services to see how your cells look (viability, debris, etc.)

    4. Adjust dissociation method and repeat test counts as needed.

    5. Set date for full experiment. Libraries are constructed.

    6. After library construction, sequencing is typically started within ~ 1 week.

**In order to set up and execute your test counts and/or single cell experiment properly, we need to meet with you and schedule your counts in advance.  We can meet nearly anytime, but please email to schedule test counts with us at least 1 week in advance as employees are already scheduled with other work this week.

Please watch our seminar with detailed information on sample prep:

https://bit.ly/3nM6BP2

  • Sample Prep Basics - 12:00
  • Tissue Prep for Visium - 34:00
  • Isolation of Nuclei from Embryonic and Adult Mouse Brain - 55:35
  • Isolation of Nuclei for RNAseq, ATACseq, and Multiome Workflows - 1:17:30
  • Customer Developed Protocols for Plant Preparation - 1:38:50

Cell Dissociation: 

  • We receive cells from nearly every source imaginable. While there may not be an exact protocol for cell dissociation of your specific tissue type, there are hundreds of protocols available on the 10x website, Worthington website, and Miltenyi website that can be adapted.

  • The Biotechnology Center Flow Cytometry Core facility offers the GentleMACS Dissociator instrument for use by University of Illinois faculty, students, and staff. You are responsible for purchasing the appropriate Miltenyi dissociation kit and tubes for your use.  There is currently no charge to use the GentleMACS Dissociator, you would check it out from Flow for use in your lab.  

  • Miltenyi makes a Tissue Storage Solution that provides for optimized storage of fresh organ and tissue samples without showing background effects like cell activation or apoptosis induction.

Cell Requirements, Tips, and Tricks:

  • Do I need replicates?:   In general, yes.  When 10x Genomics first started, people generated and published with one library per treatment. Now, reviewers are asking for replicates (3).  Our current recommendation is that you don't need replicates if you are generating preliminary data for a grant proposal.  However, if you are trying to publish the results, you should plan for 3 biological replicates.

  • Starting cell concentration should be at least 1,000 cells/ul (1M cells/ml) or more. We request 1M cells total, transported to us in a 1.5ml centrifuge tube. Only bring cells in a 1.5 or 2.0ml centrifuge tube.  You will be asked to transfer them into a 1.5ml tube if they arrive in an alternate container.  There are modified protocols for limited cells, please ask us if this is your case, we have worked with cells that could not be counted, we can make your project work too!

  • Please see this Cell Prep Guide for information on compatible buffers. Final media should not contain excessive amounts of EDTA (> 0.1mM), calcium or magnesium (> 3mM) as those components will inhibit the reverse transcription reaction. Any surfactants (Tween-20, etc) should also be avoided as they may interfere with GEM generation.

  • Cells do not need to be flow sorted prior to 10x single cell library construction. You should sort only if you need to target or remove a particular cell population, as sorting takes time, dilutes, and stresses the cells.  If you will be flow sorting first, you will need to copy both Flow Cytometry staff and DNA Services staff to set up the project dates and times.

  • Elimination of dead cells is critical. Cell viability should be >70%, but >80% is better, and over 90% is ideal. Your starting cell suspension will be counted, washed, and recounted upon arrival with live/dead measure included. If viability is below 70%, we will recommend that you repeat cell collection as background RNA levels will be high.

    • If cell suspensions cannot be obtained with >70% viability, we recommend you first try using a Flowmi 40 μm cell strainer. These filters are incredibly handy and easy to use, just put them on the tip of your 1000μl pipette tip and slowly push your suspension through to a 1.5ml tube. These will often clean up your sample nicely.

    • If cell suspensions still cannot be obtained with >70% viability, you'll need to use the Miltenyi Biotec Dead Cell Removal kit with MS columns (catalog # 130-090-101 / 130-042-201), which removes dead and dying cells.  You must purchase both the kits and the columns, linked in the previous sentence. 

    • The magnet and single-tube stand needed to use this kit can be checked out from the Biotechnology Center Flow Cytometry Core facility.

    • We also have an octo-magnet and stand in our DNA Services BL2 10x facility, 221 ERML, that you may use when you bring cells for counting.  In this case, we recommend you add the Dead Cell binding beads in your lab and use your transport time to our facility as the incubation time. Note that incubation of cells and binding beads is at room temperature, do not put your cells on ice for this step. 

  • You may also need to perform an RBC lysis step on your cells. This is the 10x answer for why and how.  If RBC lysis is needed, we recommend purchasing the RBC lysis buffer. Researchers who have tried homemade lysis buffer have significantly delayed their projects.

The numbers:

  • Prior to library setup, we should have already discussed and determined the number samples you will have, how many cells to target for each library, and how many sequencing reads needed per cell. Below you'll find some general information to help you in this decision, but please do meet with us to discuss your project rather than trying to figure it all out by yourself.

    • A library can be made from 500-10,000 cells, depending upon your project needs. Cell populations with more heterogeneity will typically require a higher total cell capture.  Most libraries we construct now target 5-6k cells per sample.

    • Doublets increase linearly with total cells, from ~0.6% at 1000 cells, to ~6% at 10,000 cells.

    • Recommended target per sample is 3,000 cells or more. Libraries capturing less than 3,000 cells have much greater variability in actual cells captured (+/- 50% or more) and lower reproducibility. Variability of actual capture at or above 3,000 cells should be ~30% or less (ie: 2,100-3,900 for a 3,000-cell target).

    • While all protocols for library preparation will be followed, due to differences in sample types submitted for 10x single cell, no guarantee is given that your cell type will behave according to typical performance.

Sequencing:

  • For 10x 3' transcriptome V3.1 NextGEM single cell kits, we recommend at least 50k sequencing reads per targeted cell. At the 50k target, you can sequence 2 libraries of ~4000 cells in 1 lane of the NovaSeq SP (~400M single reads/lane). Once your need reaches 3 lanes of SP, cost is similar to 1 lane of NovaSeq S4 2x150nt and and S4 lane is recommended.

  • Costs and sequencing options:
    • Single library: $2,330; 2-7 libraries at the same time: $2,030 each; and 8+ are $1,840 each.

    • Per lane NovaSeq SP 2x150nt: $2,400 ~400M 10X RNA reads per lane

    • Per lane NovaSeq S1 2x100nt: $4,340. ~800M 10X RNA reads per lane

    • Per lane NovaSeq S1 2x150nt: $4,700. ~800M 10X RNA reads per lane

    • Per lane NovaSeq S4 2x150nt: $5,930. ~2.5B 10X RNA reads per lane

    • For smaller projects, we can utilize single-read kits with a 28x91nt setup, as long as both lanes of the flowcell are used. This can reduce overall cost, but generates a cDNA read of 90nt, rather than 100-150nt.  

    • You can use this Excel worksheet here for pricing calculations.

Scheduling Meetings, Test Counts, and Experiments

Scheduling Test Counts:

  • There is no charge to run test counts, either independently or with assistance from DNA Services employees.  None.  All consumables for counting are also included. Please, email us to schedule these test counts, your experiment will work better and we really do want you to be successful!

  • Any new cell type should have at least one successful test count prior to the full experiment (library construction).  Most projects require just 1-2 test counts before they have achieved the viability and quality required for 10x single cell experiments.

  • Plan your test counts with us! Test count and full experiments with DNA Services employee support must be scheduled at least 1 week in advance as employees are already scheduled with other work.

  • Email this form (10x_Single_Cell_Form) to us at least 4 hours before your scheduled test count or scheduled library construction. You do not need to send in this form when using the Cell Counter independently.

    • Test counts for BL2 are performed in ERML 221 and must be scheduled with lab staff.

    • We use the Nexcelom K2 automated cell counter for 10x Single Cell libraries.  This instrument counts using both brightfield and fluorescent imaging with live/dead count as well.  It is very easy to use, flexible, and consistent.  Settings can be changed for each sample type you have and saved as your profile for easy repeat counting.

    Gaining Access to the Online Scheduler:

    •  After you have met with DNA Services staff and been trained on the Cell Counter, you may reserve times to use the Cell Counter in 334 ERML. The Cell Counter in 334 is provided for independent use, but we are present to help if you run into troubles.

    • The Cell Counter in 334 ERML is for BL1 samples only.  The DNA Services unit is a BL1 facility and you will be breaking many rules by bringing BL2 into our facility, as well as banning yourself from all future use.  Thanks for understanding and keeping us all safe.

    • When using the on-line scheduler for independent cell counting, make sure you schedule the appropriate amount of time for the number of samples you have.

      • 1-2 samples - 1-2 hours

      • 3-4 samples - 1.5-3 hours

      • 4+ samples - 2-3 hours

    • Email this very simple form (10x_Single_Cell_Scheduler_Access_Form) to ngsequencing@illinois.edu. You will receive an email when you have access to login to the scheduler. This scheduler is only used to schedule BL1 test counts. Any test counts where you will need assistance, have BL2 work, and/or full experiments (ie: the day you bring the cells for us to make the libraries) must be scheduled by contacting ngsequencing@illinois.edu.

    If your samples are BL2, you MUST contact ngsequencing@illinois.edu to schedule test counts!!

    The Online Scheduler:

    Instructions for using the 10x Single Cell Scheduler are here.

    You can access the 10x Single Cell Scheduler here.

     

    Recent Single Cell Publications from on campus:

    •Single cell RNA-sequencing reveals the complete temporal sequence of transcription factors that pattern Drosophila medulla neuroblasts. Hailun Zhu, Sihai Dave Zhao*, Alokananda Ray, Yu Zhang and Xin Li*.  In preparation.  

    •Single-cell analyses of the corneal epithelium: Unique cell types and gene expression profiles. Surabhi Sonam, Sushant Bangru, Kimberly J. Perry, Auinash Kalsotra, Jonathan J. Henry. bioRxiv 2020.08.06.240036.

    https://doi.org/10.1101/2020.08.06.240036

    •Meta-analysis of honey bee neurogenomic response links Deformed wing virus type A to precocious behavioral maturation. Traniello, I.M., Bukhari, S.A., Kevill, J. et al. Sci Rep 10, 3101 (2020).

    https://doi.org/10.1038/s41598-020-59808-4

    •Cellular plasticity balances the metabolic and proliferation dynamics of a regenerating liver. Ullas V. Chembazhi, Sushant Bangru, Mikel Hernaez, Auinash Kalsotra

    https://doi.org/10.1101/2020.05.29.124263

    •A common pattern of influenza A virus single cell gene expression heterogeneity governs the innate antiviral response to infection. J. Cristobal Vera, Jiayi Sun, Yen Ting Lin, Jenny Drnevic, Ruian Ke, Christopher B. Brooke. bioRxiv 858373. 

    https://doi.org/10.1101/858373

    •Progesterone Receptor Serves the Ovary as a Trigger of Ovulation and a Terminator of Inflammation. Park CJ, Lin PC, Zhou S, et al.  Cell Rep. 2020;31(2):107496.

    https://doi.org/10.1016/j.celrep.2020.03.060

     


    Please contact Dr. Alvaro Hernandez, Director of DNA Services (aghernan@illinois.edu) at 217-244-3480 or Chris Wright, Associate Director of DNA Services (clwright@illinois.edu) at 217-333-4372 to discuss ways the staff can be of assistance in achieving your project goals or to receive a quote for your project, submission forms, grant support or other information needed.

    All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.