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Determining cell division symmetry through the dissection of dividing cells using single-cell expression analysis


Symmetric cell divisions give rise to two sister cells that are identical to each other, whereas asymmetric divisions produce two sister cells with distinctive phenotypes. Although cell division symmetry is usually determined on the basis of a few markers or biological functions, the overall similarity between sister cells has not been thoroughly examined at a molecular level. Here we provide a protocol to separate sister embryonic stem cells (ESCs) and to conduct multiplexed gene expression analyses at the single-cell level by using 48 ESC genes. The procedure includes the dissection of dividing, paired sister cells by micromanipulation, followed by cell lysis, reverse transcription, gene-specific cDNA amplification and multiplexed quantitative PCR analyses. This protocol can be completed in 10 d, and it can be readily adapted to other cell types that are able to grow in suspension culture.

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Figure 1: Single–sister cell separation and analysis.
Figure 2: Validation of the experimental system and single-cell assays by using DKO ESCs.

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This work was supported by funding from the ICR. We thank S. Wantuch for technical assistance, T. Chen (M.D. Anderson Cancer Center) and M. Okano (Riken) for providing J1 WT and DKO embryonic stem cells, respectively, and L. Kearney, T. Ford and N. Potter (ICR) for critical reading and discussion.

Author information

Authors and Affiliations



L.J. performed experiments, analyzed data and wrote the paper. T.S. conceived, designed and performed experiments, analyzed data and wrote the paper.

Corresponding author

Correspondence to Tomoyuki Sawado.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Sequence of all primers used in the experiment. (XLSX 49 kb)

Supplementary Table 2

Biomark qPCR expression data. (XLSX 98 kb)

Microdissection of dividing sister cells.

1 s: the micropipette is placed above the paired sister cells. 9 s: the micropipette exerts physical pressure onto the junction between sister cells. 12 s: the temperature of the air trapped in the pipette decreases. 39 s: the low pressure in the pipette traps one of the sister cells. 45 s: the cell is transferred to a glass slide (the ejection part is not shown in this movie) 53 s: the pipette returns to the original picking position. 56 s: the other sister cell is picked. Bar, 20 μm. (MOV 501 kb)

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Jasnos, L., Sawado, T. Determining cell division symmetry through the dissection of dividing cells using single-cell expression analysis. Nat Protoc 9, 505–516 (2014).

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