title: hic sequence of homo sapiens: fetal control brain diencephalon
design: Experimental protocol for DLO Hi-C was performed as previous described4. Five million cells were double cross-linked with 1.5 mM EGS (ThermoSigma, Catalog code: E3257) and 1% formaldehyde (Sigma, Catalog code: F8775) and lysed in lysis buffer (10 mM Tris-HCl (pH 8.0 at 25 C), 10 mM NaCl, 0.3% Igepal CA-630, 0.5% SDS, and complete protease inhibitor (Roche)), incubated at 60 C for 5 min, and placed on ice immediately. After incubation, the nuclei were pelleted by centrifugation at 1,000 r.p.m. for 5 min and washed them once with ice-cold PBS. A total of 310 l of ddH2O, 20 l of 20% Triton X-100, 40 l of 10 NEBuffer 2.1, and 30 l of MseI (NEB, Catalog code: R0525L, 10 units/l) was then added to the nuclei and incubated for 6 h at 37 C with rotation at 15 r.p.m. After restriction enzyme digestion, 50 l of MseI half linkers (600 ng/l), 5 l of 100 mM ATP, 20 l of T4 DNA ligase (Thermo, Catalog code: EL0011, 5 units/l), and 25 l of ddH2O were added to the 400 l of digested chromatin and mixed thoroughly. The mixture was then incubated at 25 C for 1 h with rotation at 15 r.p.m. After half-linker ligation, the nuclei were centrifuged at 4 C for 5 min at 1000 r.p.m. and washed twice with 1 ml of ice-cold PBS. The linker-ligated nuclei were gently resuspended in 200 l of 1 T4 DNA ligation buffer (Thermo) containing 0.5 units/l T4 polynucleotide kinase (NEB) and incubated at 37 C for 30 min. The 200-l reaction complexes were added to 300 l of 1 T4 DNA ligation buffer (Thermo) containing 0.5 units/l T4 DNA ligase (Thermo, Catalog code: EL0011). Ligation was performed at 20 C for 2 h with rotation at 15 r.p.m. The nuclei were centrifuged at 4 C for 5 min at 1,000 r.p.m. and resuspended in 400 l of ddH2O. Protein digestion was performed by adding 25 l of 10 mg/ml proteinase K (Sigma, Catalog code: 39450-01-6), 50l of 10% SDS, and 25l of 5 M NaCl, and the tubes were incubated for 2 h at 65 C. After incubation, an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the sample, shaken vigorously, and then centrifuged for 10 min at 14,000 r.p.m. Next, the supernatant was transferred to a new tube. This process was repeated twice. DNA was precipitated at room temperature with 5l of Dr. GenTLE Precipitation Carrier (Takara, Catalog code: 9094), 50l of 3 M sodium acetate (pH 5.2), and 555 l of isopropanol. The precipitated DNA was washed once with 80% ethanol and dissolved in 160 l of ddH2O. A total of 20 l of 10 CutSmart buffer, 10 l of SAM (NEB, Catalog code: B9003SNEB), and 10 l of MmeI (NEB,; Catalog code: R0637L, 2 units/l) was added to the 160 -l DNA sample, and digestion was performed at 37 C for 1 h. The digested DNA sample was subjected to electrophoresis in native PAGE gels. The specific 80-bp DLO Hi-C DNA fragments were excised and transferred to a 0.6-ml tube with a pierced bottom. This tube was then placed into a 1.5 -ml tube, and the gel slices were shredded by centrifugation at 14,000 r.p.m. for 10 min. A total of 400 l of TE buffer was added to the 1.5 -ml tube (to ensure that the shredded gel was fully immersed in buffer), and the mixture was incubated for 20 min at -80 C, followed by a 2-h incubation at 37 C with rotation at 15 r.p.m. Next, the shredded gel, along with the buffer, was transferred into the filter cup of a 2-ml Spin-X tube filter (Costar, Catalog code: 8160). After centrifugation, the eluate was transferred into a new 2-ml tube. A total of 4l of Dr. GenTLE Precipitation Carrier (Takara, Catalog code: 9094), 40l of 3 M sodium acetate (pH 5.2), and an equal volume of isopropanol was added to precipitate the DNA. The precipitated DNA was washed once with 80% ethanol and dissolved in 40 l of ddH2O. Next, 1.5 l of PE-adaptor1 (500 ng/l), 1.5 l of PE-adaptor2 (500 ng/l), 5 l of 10 T4 DNA ligase buffer, and 2 l of T4 DNA ligase (Thermo, Catalog code: EL0012) were added to the 40 l of DLO Hi-C DNA fragments and incubated at 16 C for approximately 30 min. 90l of AMPure XP beads (Beckman, Catalog code: A63880) was added to the ligation mixes and washed twice with 80% ethanol to remove excess Illumina sequencing adaptors. Next, 45 l of ddH2O was used to wash DNA from the beads. The eluted DNA was repaired using PreCR Repair Mix (NEB, Catalog code: M0309S) for 20 min at 37 C in a final volume of 50 l. 5-10 l of repaired DNA was used as a template and amplified for fewer than 13 cycles. The PCR product is the final DLO Hi-C sequencing library.