What are the best practices for separating tissue samples into single cells?
1. Use an appropriate dissociation protocol for the tissue: The method of tissue dissociation can affect the viability of the cell suspension, so it is crucial to choose a dissociation protocol that suits the tissue type. 2. Unbiased tissue sampling: Bias in cell type composition could be introduced during tissue sampling, affecting sequencing results. It is important to sample from the same region across replicates or samples that will be compared. If only part of a large tissue is used for single-cell sequencing, thoroughly mix the dissociated tissue/cell suspension/nuclear suspension before re-sampling to reduce bias in recovered cell types. 3. Information provided by sample QC about tissue quality and dissociation methods: Assessing the RIN of tissue samples may be a way to exclude poor-quality samples, we recommend RIN > 7; cell viability and debris can indicate inefficient dissociation or over-digestion. If large tissue chunks remain, ineffective dissociation might be the issue, and appropriate enzymes can be used for dissociation; or optimize the dissociation method (pipetting, pestle, homogenizer, stirrer, grinder), increasing the number of homogenization steps may be necessary to break up large tissues; for tissues difficult to dissociate into single cells (such as the heart), consider nuclear isolation; it is also possible to use a cell filter to remove large debris. If there is a large amount of debris, it might be due to ineffective dissociation or over-digestion. This can be filtered out using a cell filter; or if possible, perform an additional wash and spin; consider handling the tissue more gently during dissociation; and optimize digestion enzymes and time.
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