Extended Data Fig. 5: Engineered rearrangements in SK-N-DZ cells.

a, Hi-C heat maps between chromosomes 7 and 8 in SK-N-SH cells (left) and SK-N-DZ cells (right). SK-N-SH cells have an endogenous t(7;8) translocation that creates a TAD fusion event at the locus, while SK-N-DZ cells have no rearrangements at the MYC locus in wild-type cells. b, Schematic for engineering rearrangement strategy. Guide RNAs targeting a locus ~300 kb downstream from the MYC gene and Guide RNAs targeting the partner region are cloned into a vector expressing Cas9. Guides are expressed either as single guides on plasmid with different fluorescent proteins or as dual guides on a plasmid with a single fluorescent protein. Cells are sorted and plated as single cells into 96 well plates. These can then be screened by PCR over the potential breakpoint to identify engineered clones. c, Sanger sequencing of PCR products from different engineered clones. The sequences that align to chromosome 7 are highlighted in green, while the sequences that align to chromosome 8 are highlighted in purple. d, Similar to Fig. 4b, validation of the engineered t(7;8) translocation by chromosome painting. e, MYC expression in cell lines containing endogenous or engineered rearrangements at the MYC locus including the non-rearranged SK-N-DZ parent cell line (purple), engineered clones classified as “Non-activating” (light blue), engineered clones classified as “MYC-activating” (dark red), Neuroblastoma cell lines with endogenous MYC rearrangements (green), and non-Neuroblastoma cell lines with MYC rearrangements (black). f, Scatter plot showing MYC expression (y-axis) and estimated MYC copy number (x-axis). g, Scatter plot showing MYC expression (y-axis) and estimated MYCN copy number (x-axis). h, Scatter plot showing MYC expression (y-axis) and MYCN expression (x-axis). i, FACS plots of mClover2 fluorescence in SK-N-DZ cells with a T2A-mClover2 reporter knocked into the 3′ end of the MYC gene (pink) and in a line derived from this MYC reporter with an engineered translocation between chromosome 1 and 8 (green). j, Heat map of chromosome 1 translocation to chromosome 8 with box showing H3K27ac ChIP-seq data over the partner region. The small inset box on the ChIP-seq track shows the enhancer targeted for deletion. k, FACS showing mClover2 fluorescence levels in the original chromosome 1 and chromosome 8 MYC reporter translocation (red) and in the same line with the targeted enhancer deletion (blue). The gate shows the region classified as “mClover2 low”. An example of the gating strategy for is also shown, including gating for single-cells and mCherry positive cells (FSC – forward scatter, SSC – side scatter, A – area, W – width). l, Percentage of “mClover2 low” cells in the control (red) and deletion (blue) cells. P-value is using Student’s two-sided T-test. m, MYC RPKM of clones with enhancer deletion on wild type allele and MYC-translocated allele. P-value is using two-sided T-test with equal variance.