Fig 2: Endogenous DSBs are enriched at TAD boundaries and around chromatin loop anchors. (a) Distributions of the sizes of TADs identified from Hi-C datasets in NES, NPC, and NEU cells. n, number of TADs. P values are indicated above the violin plots. The violin plots extend from minimum to maximum, and the boxplots inside the violins extend from the 25th to the 75th percentile, with the horizontal bar representing the median, and whiskers extending from –1.5 × IQR to + 1.5 × IQR from the closest quartile, where IQR is the inter-quartile range. The asterisks indicate a P value less than 0.0001 (Wilcoxon test, two-tailed). ns, not significant. (b) Metaprofile of the average insulation score of TAD boundaries for each of the three differentiation stages. See Methods for how the average insulation score was calculated from the Hi-C datasets. (c,d) Fraction of TADs spanning genomic regions (1 Mb resolution) belonging to the same (c) or to a different (d) compartment type. (e) Metaprofile of the DSB density around TAD boundaries identified in NES, NPC, and NEU cells based on Hi-C data. n, number of TADs. (f,g) Metaprofiles of DSB enrichment around the upstream (f) and downstream (g) anchor site of chromatin loops identified by Hi-C in NES, NPC, and NEU cells. n, number of loops. (h) Same as in (f,g) but for DSB enrichment around CTCF factor binding motifs. n, number of CTCF motifs. (i) Fraction of TADs belonging to one of six categories: (1) Early Appearing (EA); (2) Early Disappearing (ED); (3) Late Appearing (LA); (4) Late Disappearing (LD); (5) Dynamic (D); and (6) Highly Common (HC), based on whether and when TADs disappear or appear during the differentiation of NES cells to NEU. See Methods for how the classification was performed. (j) Same as in (i) but separately for each chromosome. (k) Same as in (i) but for chromatin loops. Note that the last category (grey) is now referred to as Conserved Loop (CL). (l–n) Distributions of the DSB burden per kb in a genomic region of 50 kb around each TAD boundary in NES (l), NPC (m), and NEU (n) cells. Categories assigned as in (i). (o–q) Same as in (l–n) but for chromatin loops. In all the boxplots in (l–q), each boxplot extends from the 25th to the 75th percentile, the horizontal bars represent the median, and whiskers extend from –1.5 × IQR to + 1.5 × IQR from the closest quartile, where IQR is the inter-quartile range. Black dots, outliers.

Fig 3: Endogenous DSBs are enriched in the active (A) chromatin compartment. (a) Concordance matrix revealing high similarity between the Hi-C replicates generated from NES, NPC, and NEU cells (100 kb resolution). (b) Fraction of the genome (1 Mb resolution) belonging to the A compartment as determined by Hi-C. (c) Fraction of 1 Mb genomic regions that either belong to the same (A ? A and B ? B) or switching compartment (A ? B or B ? A) during the transition from NES to NPC. (d) Same as in (c) but for the transition from NPC to NEU. (e–g) Distributions of normalized DSB counts in 1 Mb genomic regions belonging to the A or B compartment in NES (a), NPC (b), and NEU (c) cells. CPM, DSB count per million reads calculated as number of DSBs divided by number of reads times one million. n, number of 1 Mb genomic regions in each compartment. Asterisks: P value less than 0.0001 (Wilcoxon test, two-tailed). (h–j) Same as in (e–g) but comparing genomic regions that do not change (Stable) or that switch (Changing) compartment type during the transition from NES to NEU. (k–m) Same as in (h–j) but distinguishing between A/B compartments. In all the violin plots in (e–m), the violins extend from minimum to maximum and the boxplots inside each violin extend from the 25th to the 75th percentile. The horizontal bars represent the median and whiskers extend from –1.5 × IQR to + 1.5 × IQR from the closest quartile, where IQR is the inter-quartile range. The asterisks in (e–m) indicate a P value less than 0.01 (**), 0.001 (***) or 0.0001 (****) (Wilcoxon test, two-tailed). ns, not significant.

Fig 4: Validation of the model system of human neurogenesis used in this study. (a) Timeline of 2D culture of neuroepithelial stem (NES) cell differentiation to neural progenitor cells (NPC) and neuronal (NEU) cells. The rose rectangle indicates the period during which the cells were kept in differentiation conditioning medium (see Methods). Cells were harvested at three timepoints and processed for sBLISS, RNA-Seq, and Hi-C (see Supplementary Table 1 for the list of datasets). Note that different batches of NES cells from different passages (max 5 passages apart) were used for performing multiple replicate (rep) experiments with each technique. (b) Maximum z-projections of wide-field epifluorescence microscopy z-stacks showing the expression of different markers of neuronal lineage at the same days (D) of differentiation shown in (a). Scale bars, 100 µm. (c,d) Genome-wide DNA copy number profiles (100 kb resolution) of NES and NEU cells. Each grey dot represents one 100 kb genomic bin. The black lines indicate the median Log2 ratio between the observed and expected read counts per bin along each chromosome. (e) Principal component analysis of the RNA-Seq datasets (Datasets 7–15, see Supplementary Table 1). PC, principal component. Rep, replicate. (f) Hierarchical clustering of differentially expressed genes (DEG) between NES, NPC, and NEU cells. Rep, replicate. (g–n) Enrichment of 8 of the top-10 gene ontology (GO) terms associated with the differentially expressed genes shown in (f), in each of the five clusters shown in (f) or in the remaining protein-coding genes (Background).

Fig 5: qRT-PCR analysis.Notes: Unlabeled and labeled iPSC-NPs with CZF and PLL-coated ?-Fe2O3 were analyzed 2 weeks after the onset of differentiation. (A) The expression of the neural genes NES, TUBB3, SYP, and GFAP. (B) The expression of the mDN genes FOXA2, EN1, NURR1 and TH (*P<0.05). The average expression of studied markers in the unlabeled undifferentiated control was set as zero.Abbreviations: qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; iPSC-NPs, induced pluripotent stem cell-derived neural precursors; CZF, silica-coated cobalt zinc ferrite nanoparticles; PLL-coated ?-Fe2O3, poly-l-lysine-coated iron oxide superparamagnetic nanoparticles; SYP, synaptophysin; GFAP, glial fibrillary acidic protein; mDN, midbrain dopaminergic neuron.