Single Cell 3′ End Counting mRNA Seq for up to 800 Cells of 5–10 µm

mRNA Sequencing

Cell Biology
Research and Development, Fluidigm Corporation

(...) Published on Rev A

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This small-cell high-throughput (HT) script pack enables the capture of up to 800 cells (within 5 to 10 µm diameter) and the generation of cDNA by universal amplification when used together with C1™ Single-Cell mRNA Seq HT Reagent Kit v2 and an optimized integrated fluidic circuit (IFC). The results enable 3′ end counting mRNA sequencing on Illumina MiSeq, HiSeq or NextSeq systems, thus allowing the determination of the number of transcripts present per gene within an individual cell. For Research Use Only. Not for use in diagnostic procedures.

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Protocol: Single Cell 3′ End Counting mRNA Seq for up to 800 Cells of 5–10 µmDuration (H:M): 10:00

Cell Load

Sample Prep

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Tested Primary Cells or Cell Lines

Cell Name Cell Type Source
Human T cells Human activated T cells Primary
Human T cells Human naive T cells Primary
Rat neuronal cells Rat neuronal cells Primary


We prepared small cells (such as those listed in the table above) following technical note PN 101 7557 and loaded up to 2,500 cells/uL to capture >80% single cells. From the single cells isolated using the small-cell HT script pack, we used imaging analysis to identify and analyze only viable cells. The IFC comprises two cell loading ports, meaning that two biological samples can be loaded per IFC. In our development process, we worked with T cells (AllCells) and loaded active and naive human T cells on a single IFC. We loaded naive human T cells in one inlet and human T cells activated with phytohemagglutinin and interleukin 2 (PHA/IL2) in the second inlet, isolated single cells and processed them to prepare cDNA for 3′ end counting mRNA-seq. Sequencing was performed using HiSeq 2500 v4 high-output sequencing by synthesis (SBS) chemistry. Analysis was performed using the Singular Analysis Toolset. Single-cell hierarchical clustering demonstrated clear separation of stimulated verses naive T cells. tSNE analysis showed added data depth, indicating that while the majority of naive and activated T cells show clear separation, a low percentage (0.9%) of naive T cells cluster with the PHA/IL2 group. We hypothesize that this occurs because there is a higher level of IL2 expression in 0.9% of naive T cells.

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