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. 2016 Oct 1;5:e20337. doi: 10.7554/eLife.20337

Figure 2. Stem cell origin does not substantially impact on activity-dependent gene responsiveness.

(A) Example immunofluorescence pictures of Mus-ESCCORT-neurons stained for neuronal markers Tuj1 (upper) and Reelin (lower). Note also absence of Nestin staining (upper), a marker of undifferentiated neural precursor cells. Scale = 20 µm. (B) Example trace of a burst of action potentials (APs) induced in Mus-ESCCORT-neurons by current injection (see Materials and methods). (C) Example trace illustrating spontaneous TTX-sensitive AP firing. (D) Example trace illustrating spontaneous TTX-sensitive EPSCs, as well as TTX-insensitive, CNQX-sensitive miniature EPSCs (also see inset; scale bar: 20 pA, 5 ms). Activity returned upon wash out of TTX and CNQX (not shown). (E,F) Correlation of KCl/FPL-induced fold-change in the same 11,302 genes as in Figure 1d,e in Mus-ESCCORT-neurons vs. DIV10 (E) or DIV4 (F) Mus-PRIMCORT-neurons. (G) Correlation of KCl/FPL-induced fold-change in 11,302 ortholog pairs in Mus-ESCCORT-neurons vs. Hum-ESCCORT-neurons. (H) A connection map generated in Cytoscape (Shannon et al., 2003) illustrating the relative determination coefficients (R2) between the fold-inductions of the 11,302 genes studied in each of the three biological replicates of the experiments performed in each of the four different neuronal preparations. The thickness of the connecting line and the attractive force of the connecting nodes are both directly proportional to R2, against a background of constant inter-node repulsion. Note that all mouse neurons of differing developmental stage and origin (primary vs. ES cell) cluster strongly together, with the Hum-ESCCORT-neuronal replicates clustering away from them. (I) A connection map generated as for (H) but illustrating the relative determination coefficients (R2) between the basal expression levels (FPKM) of the 11,302 genes studied in each of the three biological replicates of the experiments performed in each of the four different neuronal preparations.

DOI: http://dx.doi.org/10.7554/eLife.20337.010

Figure 2—source data 1. Data set relating to Figure 2e–g.
DOI: http://dx.doi.org/10.7554/eLife.20337.011
elife-20337-fig2-data1.xlsx (2.6MB, xlsx)
DOI: 10.7554/eLife.20337.011
Figure 2—source data 2. Data set relating to Figure 2—figure supplement 1e.
DOI: http://dx.doi.org/10.7554/eLife.20337.012
elife-20337-fig2-data2.xlsx (1.8MB, xlsx)
DOI: 10.7554/eLife.20337.012
Figure 2—source data 3. Data set relating to Figure 2—figure supplement 1b–d.
DOI: http://dx.doi.org/10.7554/eLife.20337.013
elife-20337-fig2-data3.xlsx (1.3MB, xlsx)
DOI: 10.7554/eLife.20337.013

Figure 2.

Figure 2—figure supplement 1. Differential gene inducibility is not linked to basal levels of gene expression.

Figure 2—figure supplement 1.

(A) Normalised RNA-seq read density (FPKM) mapping to each gene in RNA extracted from control vs. KCl/FPL-treated Mus-ESCCORT-neurons is shown (n = 3 independent biological replicates). Genes whose expression was significantly altered by KCl/FPL treatment (Benjamini-Hochberg-adjusted p-value<0.05, calculated within DESeq2) are highlighted in red. B-D) Correlation of basal gene expression (Log2(FPKM)) across 11,302 ortholog pairs in Hum-ESCCORT-neurons vs. DIV4 Mus-PRIMCORT-neurons (B), DIV4 Mus-PRIMCORT-neurons (C) and Mus-ESCCORT-neurons (D). (E) For each of the 11,302 orthologous pairs, the Log2(DRI) Hum-ESCCORT-vs. DIV10 Mus-PRIMCORT-neurons (i.e. DRIs from Figure 1—figure supplement 1e) were plotted against the Log2(DBEI), where DBI (differential basal expression index) is the ratio of basal expression in Hum-ESCCORT-vs. DIV10 Mus-PRIMCORT-neurons.
DOI: http://dx.doi.org/10.7554/eLife.20337.014