Extended Data Fig. 1: CARGO targets LTR7 sequences in human embryonic stem cells.
a, A total of 15 sgRNAs targeting the conserved LTR7 sequences were designed for CARGO cloning. The predicted coverage ratio of all LTR7 loci is calculated based on the sequence similarity between sgRNA and LTR7 sequences. b, Classification of LTR7 loci based on their histone modification signature in H1 hESCs. H1 hESC ChIP-seq datasets were obtained from ENCODE data portal. c, Histone H3K27ac ChIP-seq signals of LTR7 loci in H1 hESCs and H1-derived differentiated lineages, including mesendoderm (ME), mesenchymal stem cell (MSC), neural progenitor cells (NPC), and trophoblast-like cells (TBL). The ChIP-seq datasets are generated by Epigenome Roadmap projects. P values determined by two-sided Wilcoxon signed-rank test. n = 1 biologically independent experiment. In the plots, center lines represent the median value, box limits the 25th and 75th percentiles, and whiskers denote minima and maxima (1.5× the interquartile range). d, Illustration of the predicated TF binding motifs (NANOG, KLF4, SOX3) and the CARGO sgRNA target sites across all LTR7 sequences. Color key: The darkness indicates counts of predicted sgRNA target sequences (red) and the number of predicated TF motifs (orange, purple, and green). In the heatmap (bottom), each row in gray color represents one LTR7 element, with pink color highlighting the sequences targeted by CARGO sgRNAs. The LTR7 elements are first sorted based on their histone modifications (H3K27ac, H3K9me3, H3K4me3, and other histone marks) and then by the number of sgRNA targeting sites. e, f, CARGO-BioID does not affect the expression of HERV-H RNA and its retroviral genes (e, RT-qPCR), or histone H3K9me3 and H3K27ac modifications on LTR7 DNA sequences (f, ChIP-qPCR). Data represent mean ± s.e.m. from three independent experiments. ns, not significant. P values were calculated by two-tailed Student′s test. In the box plots, center lines represent the median value, box limits the 25th and 75th percentiles, and whiskers denote minima and maxima (1.5× the interquartile range). g, h, The purified biotinylated nuclear proteins prepared from LTR7 CARGO-BioID experiment and subjected to proteomics analysis were analyzed by western blot (g) and silver staining (h). n = 3 biologically independent experiments.
