The downstream analysis was run with the Seurat (version 3.0) R package. The first step is to normalize the data by a global-scaling normalization method that normalizes, multiplies a scale factor (10,000), and log-transforms the feature expression. Next, features were selected to exhibit high cell-to-cell variation in the aME dataset by Seurat “FindVariableGenes” command with the following parameters: mean.function equal to ExpMean, dispersion.function equal to LogVMR, x.low.cutoff equal to 0.02, x.high.cutoff equal to 8, and y.cutoff equal to 0.5. The number of aME feature is 4844, accordingly. After a linear scaling processing, aME was used for the calculation of the PCs using Seurat’s “RunPCA” command. The final decision on the number of 3D t-distributed stochastic neighbor embedding (tSNE) transformation was made by JackStraw statistics with a sharp drop-off in significance. The statistics were estimated with 100 replicate samplings. The proportion of the data that were randomly permuted for each replicate was set to 1%. Differentially expressed features in each cluster could be calculated with “FindAllMarkers” with the threshold set to 0.25. Finally, we visualized marker expression by DoHeatmap, VlnPlot and FeaturePlot. The molecular signatures of neural stem cells (NSCs) and ependymal cells were identified by reanalyzing the published dataset from cells lining SVZ using Seurat R package19. The shared molecular markers among NSCs, ependymal cells and tanycytes were analyzed with OmicsBox software 1.4.

To systematically analyze the mitotic activity of cells in ME, we selected cell cycle-related genes for gene expression analysis as previously described49, including 43 S-phase genes (Mcm5, Pcna, Tyms, Fen1, Mcm2, Mcm4, Rrm1, Ung, Gins2, Mcm6, Cdca7, Dtl, Prim1, Uhrf1, Mlf1ip, Hells, Rfc2, Rpa2, Nasp, Rad51ap1, Gmnn, Wdr76, Slbp, Ccne2, Ubr7, Pold3, Msh2, Atad2, Rad51, Rrm2, Cdc45, Cdc6, Exo1, Tipin, Dscc1, Blm, Casp8ap2, Usp1, Clspn, Pola1, Chaf1b, Brip1, E2f8) and 54 G2/M-phase genes (Hmgb2, Cdk1, Nusap1, Ube2c, Birc5, Tpx2, Top2a, Ndc80, Cks2, Nuf2, Cks1b, Mki67, Tmpo, Cenpf, Tacc3, Fam64a, Smc4, Ccnb2, Ckap2l, Ckap2, Aurkb, Bub1, Kif11, Anp32e, Tubb4b, Gtse1, Kif20b, Hjurp, Cdca3, Hn1, Cdc20, Ttk, Cdc25c, Kif2c, Rangap1, Ncapd2, Dlgap5, Cdca2, Cdca8, Ect2, Kif23, Hmmr, Aurka, Psrc1, Anln, Lbr, Ckap5, Cenpe, Ctcf, Nek2, G2e3, Gas2l3, Cbx5, Cenpa).

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