GSM7610531: 3494-G; Homo sapiens; Hi-C
Hi-C libraries were prepared as described previously, with minor modifications. A total of 5-10 million fibroblasts or chorion cells were fixed in 1× PBS containing 2% formaldehyde (Sigma-Aldrich) for 10 min, with occasional mixing. The reaction was quenched with by the addition of 2 M glycine (Sigma-Aldrich), to a final concentration of 125 mM. Cells were pelleted by centrifugation (1,000 x g, 10 min, 4 °C), resuspended in 50 ul 1× PBS, snap-frozen in liquid nitrogen, and stored at -80°C. Cells were lysed in 1.5 ml isotonic buffer [50 mM Tris-HCl pH 8.0 (Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), 0.5% (v/v) NP-40 substitute (Fluka), 1% (v/v) Triton-X100 (Sigma-Aldrich), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)], on ice for 15 min. Cells were pelleted by centrifugation at 2,500g for 5 min, resuspended in 100 ul 1× DpnII buffer (New England Biolabs), and pelleted again. The pellet was resuspended in 200 ul 0.3% SDS (Sigma-Aldrich) in 1.1× DpnII buffer (New England Biolabs) and incubated at 37°C for 1 h. Then, 330 ul 1.1× DpnII buffer and 53 ul 20% Triton X-100 were added, and the suspension was incubated at 37°C for 1 h. Next, 600 U DpnII enzyme (New England Biolabs) were added, and the chromatin was digested overnight (14-16 h), at 37°C, with shaking (1,400 rpm). In the morning, 200 U DpnII enzyme was added, and the cells were incubated for 2 h. DpnII was then inactivated by incubation at 65°C for 20 min. The nuclei were harvested for 10 min at 5,000 g, washed with 100 ul 1× NEBuffer 2 (New England Biolabs), and resuspended in 125 ul 1.2× NEBuffer 2. Cohesive DNA ends were biotinylated by adding 25 ul biotin fill-in mixture [0.025 mM dATP (Thermo Scientific), 0.025 mM dGTP (Thermo Scientific), 0.025 mM dTTP (Thermo Scientific), 0.025 mM biotin-14-dCTP (Invitrogen), 0.8 U/μl Klenow enzyme (New England Biolabs)]. The samples were incubated at 37°C for 75 min, with shaking (1,400 rpm). Nuclei were pelleted by centrifugation at 3,000 x g for 5 minutes, resuspended in 300 ul 1× T4 DNA ligase buffer (Thermo Scientific), and pelleted again. The pellet was resuspended in 300 ul 1× T4 DNA ligase buffer, and 75 U T4 DNA ligase (Thermo Scientific) was added. Chromatin fragments were ligated at 20°C for 6 h. The cross-links were reversed by overnight incubation at 65°C in the presence of proteinase K (100 μg/ml) (Sigma-Aldrich). After cross-link reversal, the DNA was purified by single phenol-chloroform extraction, followed by ethanol precipitation [glycogen (Thermo Scientific) at a concentration of 20 μg/ml was used as co-precipitator]. After precipitation, the pellets were dissolved in 100 μl 10 mM Tris-HCl, pH 8.0. To remove residual RNA, samples were treated with 50 ug RNase A (Thermo Scientific) for 45 min at 37°C. To remove residual salts and DTT, the DNA was purified using Agencourt AMPure XP beads (Beckman Coulter). Biotinylated nucleotides from the non-ligated DNA ends were removed by incubating the Hi-C libraries (2 μg) in the presence of 6 U T4 DNA polymerase (New England Biolabs) in NEBuffer 2, supplemented with 0.025 mM dATP and 0.025 mM dGTP, at 20°C for 4 h. Next, the DNA was purified using Agencourt AMPure XP beads. The DNA was then dissolved in 500 ul sonication buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.1% SDS) and was sheared to a size of approximately 100-1,000 bp, using a VirSonic 100 (VerTis). The samples were concentrated (and simultaneously purified), using AMICON Ultra Centrifugal Filter Units (Merck), to a total volume of approximately 50 ul. The DNA ends were repaired by adding 62.5 ul MQ water, 14 ul 10× T4 DNA ligase reaction buffer, 3.5 ul 10 mM dNTP mix (Thermo Scientific), 5 ul 3 U/ul T4 DNA polymerase (New England Biolabs), 5 ul 10 U/ul T4 polynucleotide kinase (New England Biolabs), and 1 ul 5 U/ul Klenow DNA polymerase (New England Biolabs) and incubating at 20°C for 30 min. The DNA was purified with Agencourt AMPure XP beads and eluted with 50 ul 10 mM Tris-HCl (pH 8.0). To perform an A-tailing reaction, the DNA samples were supplemented with 6 ul 10× NEBuffer 2, 1.2 ul 10 mM dATP, 1 ul MQ water and 3.6 ul 5 U/ul Klenow (exo-) (New England Biolabs). The reactions were performed for 30 min at 37°C in a PCR machine, and the enzyme was then heat-inactivated by incubation at 65°C for 20 min. The DNA was purified using Agencourt AMPure XP beads and eluted with 200 ul 10 mM Tris-HCl (pH 8.0). Biotin pulldown of the ligation junctions was performed as described previously, with minor modifications. Briefly, 10 ul MyOne Dynabeads Streptavidin C1 (Invitrogen) beads were used to capture the biotinylated DNA, and the volumes of all buffers were decreased by 4-fold. The washed beads attached to captured ligation junctions were resuspended in 50 ul adapter ligation mixture, composed of 41.5 ul MQ water, 5 ul 10× T4 DNA ligase reaction buffer (Thermo Scientific), 2.5 ul Illumina TruSeq adapters and 1 ul 5 U/ul T4 DNA ligase (Thermo Scientific). Adapter ligation was performed at 22°C, for 2.5 h, and the beads were washed twice with 100 ul TWB (5 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20 (Sigma-Aldrich)), once with 100 ul 1× binding buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 M NaCl), and once with 100 ul CWB (10 mM Tris-HCl, pH 8.0 and 50 mM NaCl), and then resuspended in 20 ul MQ water. Test PCR reactions containing 4 ul streptavidin-bound Hi-C library were performed to determine the optimal number of PCR cycles necessary to generate sufficient PCR products for sequencing. The PCR reactions were performed using KAPA High Fidelity DNA Polymerase (KAPA) and Illumina PE1.0 and PE2.0 PCR primers (10 pmol each). The temperature profile was 5 min at 98°C, followed by 6, 9, 12, 15, and 18 cycles of 20 s at 98°C, 15 s at 65°C, and 20 s at 72°C. The PCR reactions were separated on a 2% agarose gel supplemented with ethidium bromide, and the number of PCR cycles necessary to obtain a sufficient amount of DNA was determined based on the visual inspection of gels (typically 9-12 cycles). Four preparative PCR reactions were performed for each sample. The PCR mixtures were combined, and the products were purified using Agencourt AMPure XP beads.