(A) Schematic overview of the in vitro human neural stem cell differentiation protocol. RNA was collected at the indicated time points and sequenced to identify significantly differentially expressed lincRNA human orthologs. (B) Heatmap of log2 ratios to undifferentiated (Day 0) human neural stem cells for 7 of the 20 lincRNAs selected for deletion with a significant (q<0.01; Cuffdiff2) increase in expression during differentiation. (C) RNA-Seq expression profile for linc–Brn1b across a panel of mouse tissues and cell types. (D) linc–Brn1b genomic locus and targeting strategy. (E) qRT–PCR confirmation of the genotype for both heterozygotes (+/−) and homozygous null (−/−) mutants. (F) Single-molecule RNA FISH targeting linc–Brn1b in wild-type E14.5 cortical neurospheres. (G) lacZ staining shows expression profile of linc–Brn1b at different embryonic (E13.5, E15.5 and E18.5) and early postnatal stages (P7) in linc–Brn1b+/− telencephalon. lacZ expression in neural progenitors of both ventral telencephalon (lateral ganglionic eminence, LGE and medial ganglionic eminence, MGE) (E13.5) and dorsal telencephalon (ventricular zone, VZ and subventricular zone, SVZ) (E15.5) is detected. Restricted expression in the upper cortical layers is observed in both E18.5 and P7 cortex. (H) Whole mount lacZ staining of P7 linc–Brn1b+/− brain shows distinct linc–Brn1b expression in primary somatosensory (S1) and visual (V1) cortical areas. (I) β-galactosidase immunofluorescence on coronal sections of P7 linc–Brn1b−/− cortex shows linc–Brn1b expression in layer IV of the somatosensory area (white boxes), specifically within the barrel structures (white arrows), as determined by co-staining with the upper layer markers SATB2 and CUX1. Abbreviations: LV, lateral ventricle; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; CGE, caudal ganglionic eminence; CP, cortical plate; SVZ, subventricular zone; VZ, ventricular zone; Str, striatum; M1, primary motor area; S1, primary somatosensory area; cc, corpus callosum; V1, primary visual cortex; A1, primary auditory cortex; F/M, frontal motor cortex. Scale bars: 500 µm (G), (I, upper panels); 100 µm (I, lower panels).