![]() FD-seq thus enables integrating phenotypic with transcriptomic information in rare cell subpopulations, and preserving and inactivating pathogenic samples. Second, we find that infection with the human betacoronavirus OC43 leads to upregulation of pro-inflammatory pathways in cells that are exposed to the virus but fail to express high levels of viral genes. We first apply FD-seq to analyze a rare subpopulation of cells supporting lytic reactivation of the human tumor virus KSHV, and identify TMEM119 as a potential host factor that mediates viral reactivation. Furthermore, FD-seq can detect a higher number of genes and transcripts than methanol fixation. We show that FD-seq preserves the RNA integrity and relative gene expression levels after fixation and permeabilization. Here we present FD-seq (Fixed Droplet RNA sequencing), a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, permeabilized and sorted single cells. This is because current high-throughput single-cell RNA sequencing methods are either incompatible with or necessitate laborious sample preprocessing for paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. A pilot may also help inform which experimental condition may be more apt.Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited. If planning for a large experiment and handling a new sample type for the first time, it may be useful to run a pilot first to test the experimental workflow and gain confidence before proceeding to scale up. Perform a pilot when possible before proceeding with large sample batches: Dead cell removal methods may be appropriate if starting with > X cellsĤ.Consider shortening enzymatic digestion or homogenization steps to preserve cell viability. ![]() Consider gentler handling of tissue during dissociation.Inefficient dissociation or overdigestion Filter out large fragments using a cell strainer.For tissues that are difficult to dissociate into single cells (i.e., Heart), consider nuclei isolation.Optimize dissociation method (pipetting, pestle, homogenizer, douncer, pulverization) may need to increase the number of homogenization steps to break up large tissues.Use an appropriate enzyme for dissociation.Cell viability and debris can be indicative of inefficient dissociation or over digestion.Assessing the RIN of a tissue sample may be a way to exclude poor-quality samples we recommend a RIN > 7.Some sample metrics can be informative on the quality of the starting tissue and dissociation method. Sample QC can provide information on tissue quality and dissociation method : If starting with a large piece of tissue and only using a portion of the sample for single-cell sequencing, mix the dissociated tissue/cell suspension/nuclei suspension thoroughly before subsampling to reduce bias in cell types recovered.ģ. It is important to ensure the same region(s) are sampled across replicates or samples that will be compared. This consideration is especially important for tissues structurally different like the brain and maybe less important for something more homogenous like the liver. Should I use warm or cold tissue dissociation?īias in cell-type composition can be introduced during tissue sampling, impacting sequencing results.How do I dissociate my tissue of interest?.Additionally, consider whether your cell type of interest has been known to be affected by warm enzymatic dissociation. Various resources and publications outline common dissociation techniques for respective tissue types. Tissue dissociation methods can impact the viability of the resulting cell suspension, so it's important to pick a dissociation protocol suitable for your tissue type. Use a dissociation protocol that works for your tissue: Question: What are the best practices for dissociating tissue samples into single cells?Īnswer: The following are some best practices to dissociate tissue into single cells :ġ.
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