Tissue Handling and Dissociation for Single-Cell RNA-Seq
The starting material for all single-cell protocols is a cell suspension. The particular functions and spatial distribution of immune cells generally make them easy to isolate them from the tissues where they dwell. Here we describe tissue dissociation pr
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Introduction All single-cell protocols start with a suspension of cells. For most tissues, this means that beforehand, the extracellular matrix that holds cells together has to be processed to loosen this mesh and to induce the release of cells into suspension. Depending on the tissue in question and the cells of interest, different approaches and a variety of conditions can be used to get a cell suspension. The functions of immune cells usually require them to move within and between tissues. Not surprisingly, they are quite resilient to being removed from a 3D tissue and to remain in suspension, in contrast with structural cell types, as epithelial and endothelial cells, which heavily depend on physical interactions with neighboring cells. Additionally, as immune cells do not constitute structural blocks of the tissues they reside in, they are generally easier to release. These two main features largely influence the dissociation protocols for immune cell isolation. Dissociation of tissues for single-cell protocols can be achieved by mechanical means, such as simple mashing, dicing, or slicing. For example, for lymphoid organs as the spleen and lymph nodes, most immune cell types can easily be isolated by mashing the tissue
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Felipe A. Vieira Braga and Ricardo J. Miragaia
through a cell strainer. When the cells of interest are embedded in more densely packed tissues, such as the dermis or the colonic lamina propria, enzymes that target collagen and other structural molecules must be used. The choice of enzyme for each tissue will depend on the composition of the extracellular matrices in terms of molecules such as fibronectin, different types of collagen, and accutin. In a lot of cases, dissociation protocols use a combination of both approaches to decrease the overall processing time: a mechanical step breaks up the tissue, increasing the surface area, followed by an enzymatic treatment. Addition of cell selection steps allowing for enrichment/depletion of certain cell types (e.g., dead cell removal, CD45 enrichment, flow cytometry), is common to most dissociation protocols. Despite their overall resilience, it is important to be aware that immune cell types can be affected by the harsh conditions of tissue dissociation. In single-cell RNA sequencing data of other cell types, it has been shown that enzymatic digestions can induce gene expression changes of a set of immediate-early genes, which then create artificial subpopulations in biologically homogeneous cell populations [1–3]. We have seen that similar artifacts can be detected to some extent in immune cell types such as T cells. Decreasing the time and temperature of digestions as much as possible is recommended in order to minimize these unwanted effects. Minimizing all other potential sources of stress, such as tempe
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