3 Preparation of Single Cell Suspensions for scRNA-Seq
The key to any scRNA-seq experiment is generating a healthy
representative single cell suspension from dissociated tissues or
embryos. Therefore, it is imperative to develop a tissue dissociation
protocol that properly captures single cells with minimal loss of
integrity of the cells and minimal degradation of RNA. To achieve
these goals, it is of utmost importance to minimize the time away
from a cell’s native environment while generating and handling
single cell suspensions to accurately capture a cell’s RNA identity,
before alterations can occur. The transient nature of RNA expression can potentially be fixed in time following dissociation with a
proper fixative, such as methanol, and the cells washed and rehydrated in 3Â SSC rather than PBS, because rehydration in PBS can
cause RNA degradation [16, 17]. Tissue types from various organisms and embryos are highly variable in their composition, therefore to generate a single cell suspension, different tissues require
different enzymes, temperatures, salinity, and pH. Many groups
have utilized enzymes that degrade extracellular matrix components to facilitate their dissociations. To establish a protocol, single
cell preparations should be kept consistent, because altering the
method of preparation can introduce a sampling bias. To establish
the optimal conditions our single cell dissociation protocol was
developed using a pilot study to establish the most reliable
approach and as part of that, establish that a fixative such as methanol can be used to stabilize the RNA. The pilot study helped
establish optimal scRNA-seq conditions for our system. The details
of dissociation and stabilization of RNA are too varied to be covered in this chapter, but in each case the goals outlined above
should be sought.
4 Considerations of Approach and Instrumentation Available for Library Preparation
from Single Cells
To a research group beginning a scRNA-seq project, the next big
question to ask is what platform should be used? Single cell
RNA-sequencing has rapidly evolved since it was first used in
2009 [18]. When scRNA-seq was first introduced, it involved
manually pipetting single cells into microwells and was relatively
low throughput with a considerable amount of work required per
cell. Since then, many groups have contributed to making scRNAseq cost efficient and high throughput, and today many variations
of these technologies exist. The introduction of multiplexing in
2011 [19, 20] was a major milestone where they showed many
single cells could be sequenced together when UMIs were used.
Additionally, in 2013 [21] integrated fluidic circuits, to allow for
Methods for in Vivo EMT at Single Cell Resolution
307
The key to any scRNA-seq experiment is generating a healthy
representative single cell suspension from dissociated tissues or
embryos. Therefore, it is imperative to develop a tissue dissociation
protocol that properly captures single cells with minimal loss of
integrity of the cells and minimal degradation of RNA. To achieve
these goals, it is of utmost importance to minimize the time away
from a cell’s native environment while generating and handling
single cell suspensions to accurately capture a cell’s RNA identity,
before alterations can occur. The transient nature of RNA expression can potentially be fixed in time following dissociation with a
proper fixative, such as methanol, and the cells washed and rehydrated in 3Â SSC rather than PBS, because rehydration in PBS can
cause RNA degradation [16, 17]. Tissue types from various organisms and embryos are highly variable in their composition, therefore to generate a single cell suspension, different tissues require
different enzymes, temperatures, salinity, and pH. Many groups
have utilized enzymes that degrade extracellular matrix components to facilitate their dissociations. To establish a protocol, single
cell preparations should be kept consistent, because altering the
method of preparation can introduce a sampling bias. To establish
the optimal conditions our single cell dissociation protocol was
developed using a pilot study to establish the most reliable
approach and as part of that, establish that a fixative such as methanol can be used to stabilize the RNA. The pilot study helped
establish optimal scRNA-seq conditions for our system. The details
of dissociation and stabilization of RNA are too varied to be covered in this chapter, but in each case the goals outlined above
should be sought.
4 Considerations of Approach and Instrumentation Available for Library Preparation
from Single Cells
To a research group beginning a scRNA-seq project, the next big
question to ask is what platform should be used? Single cell
RNA-sequencing has rapidly evolved since it was first used in
2009 [18]. When scRNA-seq was first introduced, it involved
manually pipetting single cells into microwells and was relatively
low throughput with a considerable amount of work required per
cell. Since then, many groups have contributed to making scRNAseq cost efficient and high throughput, and today many variations
of these technologies exist. The introduction of multiplexing in
2011 [19, 20] was a major milestone where they showed many
single cells could be sequenced together when UMIs were used.
Additionally, in 2013 [21] integrated fluidic circuits, to allow for
Methods for in Vivo EMT at Single Cell Resolution
307