Fig. 5

lincRNA TUNA Is Required for Neuronal Differentiation of mESCs

(A) qRT-PCR analysis of TUNA, Nestin, and Oct4 expression during neuronal differentiation of CCE mESCs. Total RNA was extracted on the indicated days of monolayer neural differentiation cultures, and relative mRNA levels were normalized to 18S rRNA. Data are the mean ± SD of triplicates.

(B) qRT-PCR analysis of neuronal lineage genes in CCE mESCs following shRNA-mediated silencing of TUNA. RNA was extracted 4 days after transduction, and mRNA levels were normalized to Actb.

(C) Heatmap showing hierarchical clustering of differentially expressed genes following TUNA knockdown. Shown are genes with a difference e3-fold in expression in TUNA-depleted versus control CCE mESCs. RNA was extracted on days 0, 2, 4, and 6 of neural differentiation culture.

(D) Enriched gene ontology (GO) processes of the ten gene clusters identified in (C).

(E and F) Fluorescence and bright-field micrographs of in vitro differentiated CCE mESCs transduced with control or TUNA-specific shRNA. CCE cells were transduced after 2 days of differentiation and analyzed at day 7. Upper panels show cells immunostained for Nestin (E) and Tuj1 (F). Middle panels show Hoechst nuclear staining, and lower panels show bright-field images. Scale bars, 100 µm.

(G) Brain histone H3K4me3 ChIP-seq analysis (UMMS Brain Histone [Akbarian/Weng] UCSC track, GRCh37/hg19) of the human TUNA ortholog (LINC00617) in neuronal and nonneuronal nuclei collected from the prefrontal cortex of a 4.7-year-old male and a 69-year-old female.

(H) Knockdown of human TUNA (LINC00617) abolished neural differentiation in human. The monolayer culture method was performed in H9 hESCs. Lentiviral infection was performed on day 3 upon neural induction. Neural progenitor cells were immunostained for Pax6 at day 8. Scale bars, 50µm. See also Fig. S5 and Table S4.