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Analysis of allelic expression patterns in clonal somatic cells by single-cell RNA–seq

Abstract

Cellular heterogeneity can emerge from the expression of only one parental allele. However, it has remained controversial whether, or to what degree, random monoallelic expression of autosomal genes (aRME) is mitotically inherited (clonal) or stochastic (dynamic) in somatic cells, particularly in vivo. Here we used allele-sensitive single-cell RNA–seq on clonal primary mouse fibroblasts and freshly isolated human CD8+ T cells to dissect clonal and dynamic monoallelic expression patterns. Dynamic aRME affected a considerable portion of the cells' transcriptomes, with levels dependent on the cells' transcriptional activity. Notably, clonal aRME was detected, but it was surprisingly scarce (<1% of genes) and mainly affected the most weakly expressed genes. Consequently, the overwhelming majority of aRME occurs transiently within individual cells, and patterns of aRME are thus primarily scattered throughout somatic cell populations rather than, as previously hypothesized, confined to patches of clonally related cells.

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Figure 1: The vast majority of aRME in primary mouse fibroblasts is dynamic.
Figure 2: Scarce clonal aRME in weakly expressed genes.
Figure 3: Dynamic and clonal aRME in human T cells.
Figure 4: Cellular size and cell cycle phase affect the degree of dynamic aRME.

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Gene Expression Omnibus

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Acknowledgements

We thank D. Edsgärd for assistance in handling sequence data and members of the Sandberg laboratory for their input. J.M. was supported by a Human Frontiers Science Program Long-Term Fellowship (LT-000231/2011-l), and the work was supported by grants from the Swedish Research Council, the European Research Council (648842), the Swedish Foundation for Strategic Research, the Swedish Cancer Society, the Karolinska Institute, Tobias Stiftelsen, the Strategic Research Programme in Stem Cells and Regenerative Medicine at Karolinska Institutet (StratRegen), Knut och Alice Wallenbergs Stiftelse, and Torsten Söderbergs Stiftelse.

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Authors

Contributions

B.R. designed mouse experiments, derived and sequenced the transcriptomes of mouse cells, performed computational experiments, prepared figures and tables, and wrote the manuscript. J.E.M. designed human experiments, performed FACS, sequenced the transcriptomes of human T cells, and analyzed TCR sequences. D.R. performed computational experiments and prepared figures. Q.D. designed mouse experiments and derived mouse cells. P.J. performed cell cycle classification. J.M. designed human experiments and performed FACS. J.F. designed human experiments. R.S. designed mouse experiments, supervised the work, and wrote the manuscript.

Corresponding author

Correspondence to Rickard Sandberg.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–22 and Supplementary Note. (PDF 10057 kb)

Supplementary Table 1

List of RNA–seq libraries. (XLSX 114 kb)

Supplementary Table 2

Genes escaping X inactivation in primary mouse fibroblasts. (XLSX 104 kb)

Supplementary Table 3

Imprinted genes detected by single-cell RNA-seq and bulk RNA-seq. (XLSX 42 kb)

Supplementary Table 4

Genes with clonal aRME in primary mouse fibroblasts. (XLSX 66 kb)

Supplementary Table 5

GO analysis of clonal aRME genes identified in mouse primary fibroblasts. (XLSX 59 kb)

Supplementary Table 6

Genes with clonal aRME in ex vivo–expanded human T cells. (XLSX 64 kb)

Supplementary Table 7

GO analysis of genes differentially expressed in fibroblasts with large and small diameters. (XLSX 98 kb)

Supplementary Table 8

GO analysis of genes positively correlated with the total amount of mRNA per fibroblast cell. (XLSX 219 kb)

Supplementary Table 9

GO analysis of genes negatively correlated with the total amount of mRNA per fibroblast cell. (XLSX 232 kb)

Supplementary Table 10

Genes differentially expressed in large and small fibroblasts. (XLSX 63 kb)

Supplementary Table 11

Gene expression level correlated with the total number of mRNA molecules per fibroblast cell. (XLSX 1527 kb)

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Reinius, B., Mold, J., Ramsköld, D. et al. Analysis of allelic expression patterns in clonal somatic cells by single-cell RNA–seq. Nat Genet 48, 1430–1435 (2016). https://doi.org/10.1038/ng.3678

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