Introduction to single cell immune sequencing
Unlike bulk repertoire analysis, single cell sequencing maintains the cognate pairing between receptor chains in individual immune cells. Our single cell repertoire sequencing service, called iPair, was—to our knowledge—the first consumer-facing service to capture paired TCR alpha-beta or BCR IgH-KL chains. In preparation for amplification and sequencing, cells of interest need to be identified, potentially sorted through either magnetic bead separation or staining, and then single-cell plated. Each of these steps can be performed either in-house by your laboratory or as a service in ours.
To clarify, we use the term “sort” to refer to the separation of a subset of specific cells (such as CD4+) from the bulk population of cells (such as PBMC). The term “plate” is used to reference the physical deposition of a single cell into a single well of a 96-well plate. All cells will need to be plated for iPair services, but not all cell samples will require cell sorting; this will depend on your individual research goals.
There are several ways to initiate an iPair project, and these are outlined below.
Cell sorting and plating at your site
If you have access to FACS with single-cell capability, you can single-cell sort and/or plate at your site using the iPair Plating Kit and then send the plate(s) to us for service. It is highly recommended to perform a sorting validation prior to your first real sample submission. We will perform a compatibility check of your sorter and method with our process by amplifying a portion of the plate; no sequencing is performed unless specifically requested. This step ensures both the person sorting and the sorter are validated for future submissions. Any deviations must be re-validated.
Cell sorting only at your site
Alternatively, you can magnetically sort or use FACS to bulk sort many cells in one Eppendorf tube, store the cells in Qiagen’s RNAprotect Cell Reagent (cat. no. 76526), and ship to us at 4°C. When doing this, bulk sort as usual, spin the cells down, remove all supernatant, and resuspend fully in 300-500 uL of RNAprotect Cell Reagent. Cells will be single-cell plated at iRepertoire’s service laboratory upon receipt.
Cell sorting and plating at our site
If you lack sorting capability or would prefer these steps to be handled by iRepertoire, a project consultation is required. We can handle cryopreserved bulk populations of cells and single-cell sort the cells of interest for you. Charges for this vary upon project details and sample handling requirements.
For more information, please visit our iPair page.
Recommendations for cell handling
The following are the cell handling recommendations we’ve found to improve cell viability (and thus downstream success) during FACS sorting and/or plating.
- A live-dead stain is highly recommended to increase single-cell analysis success rates.
- A nozzle or chip size of 100 or 130 microns is recommended as it may help reduce stress on the cells.
- Lowering the sample pressure can also reduce stress on the cells. If instrument supports reducing sample pressure, please do so. For Sony SH800 operation, in-house sample chamber pressure is set to 4 on an arbitrary scale of 10.
- For iPair validation plates, we suggest using the Jurkat cell line (Sigma Aldrich cell line 88042803)
- If plating at your site, please perform a plate alignment prior to beginning the experiment.
- No intracellular staining. We have found that cells that have undergone intracellular staining have an extremely low amplification success rate for iPair.