Optimized Hi-C Library Generation

Generation of Hi-C libraries with low cell numbers was optimized according to a previous protocol^{11 (link)}. Briefly, 100–600 embryos were cross-linked with 1% formaldehyde for 40 min using vacuum infiltration. Isolated embryo nuclei were digested with 80 U of DpnII (catalog no. R0543L; New England Biolabs) at 37 °C for 5 h. Restriction fragment overhangs were marked with biotin-labeled nucleotides. After labeling, chromatin fragments in proximity were ligated with 4,000 U of T4 DNA ligase for 6 h at 16 °C. Chromatin was reverse-cross-linked, purified and precipitated using ethanol. Biotinylated ligation DNA was sheared to 250–500-base pair (bp) fragments, followed by pull-down with MyOne Streptavidin T1 Dynabeads (catalog no. 65602; Thermo Fisher Scientific). Immobilized DNA fragments were end-repaired, A-tailed and ligated with adapters. Fragments were then amplified with the Q5 High-Fidelity 2X Master Mix (catalog no. M0492L; New England Biolabs). Hi-C libraries were sequenced on the Illumina HiSeq X10 platform (paired-end 2 × 150-bp reads).