The DynaSCOPE® Single Cell RNA Dynamic Library Kit employs chemical labeling to discriminate nascent mRNA allowing to distinguishing newly transcribed and pre-existing RNA within single cells.
The accurate measurement of freshly synthesized and long-lasting mRNA might help to understand immediate regulatory dynamics in tissue as well as developmental, environmental changes, drug treatments and infection mechanisms from an experimental and bioinformatical perspective.
During the DynaSCOPE workflow, a nucleotide analogue (uracil-analogue) is incorporated into freshly synthesized transcripts during transcription, but not into static transcripts. The incorporated uracil-analogue is converted into cytosine analogue and later recognized as cytosine (C), while the unlabeled uracil in stable transcripts is converted into thymine (T). Nascent and long-lasting transcripts can then be differentiated by bioinformatic analysis provided by the CeleScope® software.
(A) Two mice at different ages were abdominally injected with labelling reagent followed by DynaSCOPE library preparation. (B) Cell and sample type annotation are shown in the UMAP plot . (C) UMAP plot shows transcriptionally active cell clusters with shades of red indicating the transcriptional synthesis rate.
Dynamics of the transcriptomic landscape of mouse bone marrow cells was analyzed using the DynaSCOPE® Single Cell RNA Dynamic Library Kit. Following cell labeling, an elevated proportion of nascent RNA to total transcript was observed during a 2-hour period, highlighting the critical role of incorporating the time dimension into experimental design.
Expression profile of the very highly enriched genes in lung tissue show different transcriptional behaviors in various clusters that are not visible from conservative single-cell RNA-seq data.
S4U labelling can be performed in vitro or in vivo
Full instruments-free workflow
PythoN: tissue dissociation NEO: single cell processing
Suitable labelling time depends on the tissue used
choose between SD and HD chip depending on required capture rate
The selection of labeling time to effectively distinguish RNA synthesis rates under various conditions is contingent upon sample types and the inherent RNA synthesis rate in different cells. It is recommended to incubate cells for a duration ranging between 0.5 and 4 hours. For most sample types, a standard duration of 2 hours is typically recommended.
For optimal metabolic labeling, it is advised to utilize DMEM medium supplemented with 20% FBS.
The suggested total number of cells for metabolic labeling is in the range of 1 to 2 million (1-2 × 10^6).
The effect on cell viability varies with cell types; some cells may experience a 5-10% decrease in viability after culture. It is imperative to maintain high cell viability, aiming for approximately 80%. The total number of living cells should exceed 40,000 after incubation for subsequent steps.
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