What cell preparation protocol is recommended to obtain optimal C1 data when using TraCeR analysis?
The successful detection of T cell receptor sequences will depend on the amount of the relevant transcript available for isolation, and this will vary depending on the subtypes of T cells captured by the C1 IFC and on the sample preparation protocol. Stubbington et al. (Nature Methods, 2016), who developed TraCeR, used freshly isolated mouse splenic T cells and were able to detect a variety of subtypes, including activated proliferating T cells and central and effector memory T cells. Detecting transcripts in single, naïve T cells may be difficult due to their quiescent nature and low transcription rates. Recommended sequencing depth is 1 million reads per T cell.
The sample handling protocol will also influence the amount of transcript available for amplification. Take the following precautions to minimize cell stress:
- Avoid cryopreservation.
- If cryopreserved samples must be used, implement a protocol that has been optimized for cell recovery.
- Minimize the amount of time cells are held either post-harvest or post-thaw.
- Conduct antibody labeling or other enrichment steps at 4 °C.
- If you are pre-enriching by flow sorting, use a low-pressure and larger nozzle (100 µm) for reduced shear stress to the cells.