## 3 Results
### Co-Expression Analysis of Coding and Non-Coding Transcripts
As a result, 3 co-expression modules were constructed and were shown in different colors. The turquoise module was the largest one with 89 genes, followed by the blue with 62 genes and the brown with 46 genes. (**B**) Heatmap showing correlation between gene modules and conditions (e.g., Nichoid-growth NPCs and standard floating conditions). Different colors are related to different correlation values. For further analysis we focused our attention on turquoise and blue modules. (e.g., _Gm44773, Gm29206, Gm26802, Gm26892, 2310081003 Rik, Gm29671, A330087D11 Rik, C230057A21 Rik, E330013P04 Rik)_, represented in light blue, and two antisense (e.g., _Nr6a1 os_, 4933432K03 Rik), represented in pink, interact with each other and with 37 coding genes (Figure 3A). Interestingly, all coding genes involved in the interaction are down-regulated and are thus represented in green according to their fold change (Figure 3B). Moreover, with respect to small ncRNAs, represented in blue, _Snora28_ co-interacts with _Gm24494_ and _Snora61_ in a smaller network, forming a non-coding only network (Figure 3B). Remarkably, none of the lncRNAs that emerged in the WGCNA analysis (in light blue or pink respectively in Figure 3), except for _Gm16892_ and _Gm28592_, have been functionally characterized yet. Figure 3: Study of investigated ncRNAs networks. On the basis of the turquoise and blue gene modules, two networks were constructed via Cytoscape. Both networks display coding genes represented in green or orange according to \(\mathrm{llog_{2}FC1}\), \(\mathrm{lincRNAs}\) light blue, antisense RNAs in pink, snoRNAs in dark blue. (**A**) The first network was obtained from the turquoise module. Here, 7 lincRNAs and 1 antisense, highlighted respectively in light blue and in pink, interact with each other and with 58 coding genes. All coding genes involved in this interaction are up-regulated and are thus represented in orange according to their fold change. (**B**) The second network was obtained from the blue module. Here, 9 lincRNAs, represented in light blue, and 2 antisense, represented in pink, interact with each other and with 37 coding genes. All coding genes involved in the interaction are down-regulated and are thus represented in green according to their fold change. Moreover, with respect to small ncRNAs, represented in dark blue, _Snora28_ co-interacts with _Gm24494_ and _Snora61_ in a smaller network, forming a non-coding only network. On the basis of the turquoise and blue modules we performed a functional enrichment analysis via the g:Profiler web tool (see M&M section), thus considering not only the different gene modules but also the up and down regulated coding genes that emerged (Figure 3A,B), in order to better investigate the possible functional implications of lncRNAs and small ncRNAs in Nichoid-expanded NPCs. A significant deregulation was observed in all categories analyzed, which include Gene Ontology (GO), KEGG, Reactome, and WikiPathways. Specifically, we identified 184 significant pathways for molecular function (MF), 1428 significant pathways for biological processes (BP), and 130 significant pathways for cellular component (CC). Pathway analyses highlighted 59 significant pathways for KEGG, 151 significant pathways for Reactome, and 16 significant pathways for WikiPathways when considering the turquoise module (Table S2). On the other hand, the down-regulated coding RNAs found in the blue module highlighted 174 significant pathways for MF, 1193 significant pathways for BP, and 73 significant pathways for CC, along with 25 significant pathways for KEGG, 59 significant pathways for Reactome, and 14 significant pathways for WikiPathways (Table S2). Functional Enrichment Analysis of Genes in Co-Interacting Modules Predicts Functions for Non-Coding RNAs in Gene Ontology  
The GO Term analysis in MF highlighted 184 significantly deregulated pathways for the turquoise module and 174 for the blue one and the top 10 ranked by significance are displayed as dot-plot graph respectively in Figure 4A,B.