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In "RNA velocity of single cells", La Manno et al. look at ratios between spliced and unspliced mRNA as a way of estimating the velocity of a cell through transcriptome space.

In checking for a universal steady-state spliced-unspliced ratio, they write "We found that the steady-state behaviour of most genes across a wide range of cell types was consistent with a single fixed [ratio between spliced and unspliced] (Extended Data Fig. 3a–c). However, 11% of genes showed distinct slopes in different subsets of tissues (Extended Data Fig. 3d, e), suggesting tissue-specific alternative splicing (Extended Data Fig. 3f) or degradation rates."

Tissue-specific alternative splicing or degradation rates certainly could account for these patterns, but how do La Manno et al. rule out that some tissues are simply not in a steady state? The figure they cite (screenshotted below) uses data from the Tabula Muris adult mouse cell encyclopedia. In an adult mouse, not all cells would not be expected to be in steady state -- for example, T-cell progenitors from the thymus and epithelial cells in many organs undergo continual replenishment.

enter image description here enter image description here

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If cells aren't in a steady state then one wouldn't expect to see the results in (e), where there are two slopes. One would instead expect to see results more like Plekha3 in panel (c), where there's more variability around the the steady state line (or perhaps no steady state line at all).

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  • $\begingroup$ Could you explain why that is that case? For example, couldn't you get two slopes as follows? Suppose that keratin 17 increases during development of multiple epithelial lineages, but is constant in non-epithelial cell types. For epithelia, there would be an excess of unspliced transcripts due to the portion of the population that is developing. The epithelia would have a line with a high slope and the rest would have a line with a low slope. $\endgroup$ Sep 24 '18 at 14:09
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    $\begingroup$ The difference between developing and differentiated is a gradient. $\endgroup$
    – Devon Ryan
    Sep 24 '18 at 14:17
  • $\begingroup$ I think I understand. So if cells are on average less differentiated in intestinal epithelia and more differentiated in tracheal epithelia, you'd expect them not to fall a line of the same slope? $\endgroup$ Sep 24 '18 at 14:25
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    $\begingroup$ You wouldn't expect undifferentiated cells to fall on any particular line, since that's a broad spectrum of states. They may have some line that they tend to fall on, but you'd then expect to see a lot of noise between that and the "differentiated line", due to various levels of differentiated status. $\endgroup$
    – Devon Ryan
    Sep 24 '18 at 14:28
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In addition to Devon Ryan's data-driven answer, I've found a clue in the text. La Manno et al have not used the full Tabula Muris: the figure legend indicates that they used only terminally differentiated cells.

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