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. 2017 Sep 13;7:11458. doi: 10.1038/s41598-017-11574-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Experimental design. (a) NES cell differentiation: bright field images of NES cells and differentiated neurons. (b) Timeline of the experiment. NES cells were plated and kept in the presence of FGF and EGF for two days. Growth factors were removed and cells were differentiated for 38 days. Caffeine was added and RNA was collected after 0 h, 1 h, 3 h and 9 h. The samples were then used for CAGE sequencing and differential expression analysis. (c) Phenotype of differentiated cells. After 38 days, cells were fixed and stained for the neuronal markers MAP2 and NeuN and the nuclear marker DRAQ5. Untreated cells are shown.