Single Cell Projects

Our genomics core has performed hundreds of single cell captures since implementing 10x Genomics techniques in our core and we are happy to work with you to figure out the best way to set up your project. To ensure we have the necessary reagents and staff available to cover your captures, we ask that our clients follow our scheduling procedure below so we can ensure consistent coverage of everyone's projects.

Scheduling

If you need to schedule captures for fresh (live) cells, please follow the steps below:

  1. Email us at least one month in advance with your requested date/time and number of captures. If you have unavoidably short timelines, please call us to discuss your project.
  2. Once your capture has been confirmed you will receive a calendar invite for the specified date and time. If you have not received a calendar invite, the capture has not been scheduled.
  3. Fill out our online project submission form at least 24 hours in advance of your capture. Samples will not be accepted without a completed project form.
  4. Keep us updated on the day of the capture by email in case there are any changes to the timeline (please email all staff members included on the calendar invite).

Sample Preparation Information

For best results, single cell RNAseq captures should be performed on freshly collected cells so it is important to schedule these experiments in advance. If you have not previously done any single cell experiments with us, please contact us to discuss your project.

  • Cells should be provided as single cell suspensions in a compatible media. See the 10x Genomics Cell Prep Guide for more details. We frequently receive cells in RPMI + 10% FBS for 10x captures.
  • If your cell preparation contains red blood cells, consider using a RBC lysis protocol. The 10x device will capture all types of cells that are loaded.
  • Please provide us with either a cell count from a hemocytometer or the number of cells that you have sorted to give us an idea of what is in the sample.
  • Once we receive the samples, we will perform a count and check the viability if possible (for samples with at least 20000 cells). If the viability and count indicate a high likelihood of an unsuccessful capture, we will contact you to discuss options. Please make sure that you provide us with a contact number so that we can discuss any issues immediately.
  • Handle cells gently (no rough pipetting or harsh vortexing) and keep them on ice at all times they are not being actively worked with.

If you do not have an optimized protocol for dissociating your freshly collected tissue into single cell suspensions or have biobanked frozen tissues, we recommend performing single nucleus sequencing. Isolation of viable single cells from thawed tissue is challenging. For snap freezing, transfer your tissue into an empty cryovial and submerge this in liquid nitrogen. The frozen tissue can be provided to us at your convenience.

The core can perform the isolation of nuclei from snap frozen tissue. We are able to work with a variety of tissue types but require 20 mg – 50 mg of tissue to obtain enough nuclei for capture. If possible, before snap freezing, dissect the tissue into pieces that are no larger than 50 mg. In your sample manifest, please provide the weight of each of the tissue samples that you are submitting.

Fixation is compatible with the 3’ or 5’ Universal Gene Expression and Flex Gene Expression assays. The RT-based single cell Universal Gene Expression assays are compatible with cells that have undergone DSP-methanol fixation, and the Flex Gene Expression assays are performed on cells fixed with paraformaldehyde. The core currently accepts fixed cells and nuclei prepared following the "CG000782 Fixation of Cells & Nuclei for GEM-X Flex Gene Expression" or the "CG000776 Cell Fixation Protocol for GEM-X Single Cell 3’ & 5’ Assays" Demonstrated Protocols from 10x Genomics. Please discuss your batching and multiplexing strategies with us once you have completed the collection and fixation of all required samples.

The sample multiplexing with hashtags workflow is not fully supported by 10x Genomics, and there are limited official resources on this multiplexing modality. Please contact us if you are unfamiliar with these reagents or are working with them for the first time and especially if you intend to sort your samples and/or have a low number of cells. The Genomics Core does not perform the staining of cells with hashtag antibodies, and this must be completed before you provide the samples to us.